Accepted Manuscript Title: A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids Author: Clara John Philipp Werner Anna Worthmann Katrin Wegner Klaus T¨odter Ludger Scheja Sascha Rohn Joerg Heeren Markus Fischer PII: DOI: Reference:
S0021-9673(14)01655-0 http://dx.doi.org/doi:10.1016/j.chroma.2014.10.064 CHROMA 355942
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
5-9-2014 20-10-2014 21-10-2014
Please cite this article as: C. John, P. Werner, A. Worthmann, K. Wegner, K. T¨odter, L. Scheja, S. Rohn, J. Heeren, M. Fischer, A liquid chromatographytandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.10.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A liquid chromatography-tandem mass spectrometry-based method for the simultaneous de-
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termination of hydroxy sterols and bile acids
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Clara John1*, Philipp Werner2*, Anna Worthmann1, Katrin Wegner2, Klaus Tödter1, Ludger Scheja1,
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Sascha Rohn2, Joerg Heeren1# and Markus Fischer2#
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* These authors contributed equally
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Eppendorf, Martinistr. 52, 20246 Hamburg; 2Hamburg School of Food Science, Institute of Food
Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-
Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany
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#
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Markus Fischer, Hamburg School of Food Science, Institute of Food Chemistry, University of Ham-
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burg, Grindelallee 117, 20146 Hamburg, Germany. Phone: +49(0)40-42838-4342, Email: mar-
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[email protected]
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Joerg Heeren, University Medical Center Hamburg-Eppendorf, Dept. of Biochemistry and Molecular
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Cell Biology, Martinistraße 52, 20246 Hamburg, Germany. Phone: +49(0)40-4710-54745, Email:
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[email protected]
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Corresponding Authors:
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Abstract
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Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators
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of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid
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species requires powerful analytical tools to quantify these structurally and chemically similar ana-
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lytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based
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method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as
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their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols
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and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase.
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The current method features a simple extraction protocol validated for blood plasma, urine, gall blad-
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der, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization proce-
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dures. The total extraction recovery for representative analytes ranged between 58-86 % in plasma, 85
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% in urine, 79-92 % in liver, 76-98 % in adipose tissue, 93-104 % in feces and 62-79 % in gall blad-
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der. The validation procedure demonstrated that the calibration curves were linear over the selected
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concentration ranges for 97 % of the analytes, with calculated coefficients of determination (R2) of
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greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched
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Western type diet illustrated that the protocol described here provides a powerful tool to simultane-
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ously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the
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enterohepatic circulation.
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1. Introduction
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Sterols are apolar lipids formed by four fused rings resulting in an inflexible core. While plants syn-
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thesize diverse phytosterols, cholesterol is the most abundant sterol synthesized in animal cells. Its
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chemical structure allows cholesterol to serve as a component of membranes, facilitating membrane
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fluidity, as well as a precursor of steroid hormones and other signaling molecules. In carnivores, cho-
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lesterol supply is provided either by diet or by de novo synthesis [1,2].
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High intracellular cholesterol levels are cytotoxic and a disturbed cholesterol metabolism observed in
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patients with hypercholesterolemia is causally associated with the development of cardiovascular dis-
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ease [3]. An important way to get rid of excess cholesterol is the biliary excretion of either non-
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esterified cholesterol or its hydrophilic derivatives, the bile acids (BA) [4,5]Besides their role as in-
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termediates in the BA synthesis pathway, hydroxy sterols are well known agonists for the transcription
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factor liver X receptor and the estrogen receptor α (ERα), thereby regulating systemic lipid and energy
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metabolism [6] as well as inflammatory processes known to be important for the development and
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progression of chronic metabolic diseases such as cardiovascular disease, type 2 diabetes mellitus and
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cancer [7-9]. In a multi-step reaction catalyzed by enzymes located in the liver [5,10-14], hydroxy
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sterols are converted into primary bile acids (PBA). In humans, cholic acid (CA) and chenodeoxy-
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cholic acid (CDCA) are the main primary bile acids whereas CA and muricholic acid (MCA) pre-
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dominate in mice [5]. Further conjugation to taurine (predominant in rodents) or glycine (predominant
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in humans) increases hydrophilicity. Via bile ducts BA reach the intestine where they facilitate absorp-
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tion of dietary fats. Secondary bile acids (SBA) arise from action of the gut microbiota [4]. Besides
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their function in fat absorption, BA have signaling functions, acting as agonists and antagonists of the
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transcription factor farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (GBAR1
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also known as TGR5). These receptors mediate bile acid, lipid, and glucose homeostasis as well as
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energy expenditure [6,15-18]. The multifunctional presence of hydroxy sterols and bile acids in differ-
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ent biochemical pathways shows that their quantitative observation in different biofluids and organs is
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of great scientific relevance, especially with respect to the study of chronic inflammatory metabolic
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diseases. Unfortunately, there are very few analytical methods for the determination of hydroxy sterols
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and bile acids in one analytical approach. A bottleneck to the analysis of hydroxy sterols and bile acids
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is the chromatographic separation prior to analysis via mass-spectrometric detection. As many of the
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analytes are isobaric and very similar in their structure, no selective multiple reaction monitoring-
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(MRM) transitions can be obtained. This results in the need for chromatographic baseline separation.
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In the past, this problem has often been solved by determining very similar compounds as a sum pa-
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rameter or via gas chromatography (GC)-MS-approaches [19]. GC-based applications are able to sepa-
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rate positional isomeric sterol compounds but need further sample preparation steps including complex
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derivatization techniques. As sterols and their derivatives can have very different biochemical func-
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tions, these procedures are not purposeful for some scientific research questions [20,21].
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In this work, our aim was to provide a method for the quantitative determination of 34 sterols in bio-
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logical matrices using high performance liquid chromatography electrospray ionization triple quadru-
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pol tandem mass spectrometry (HPLC-ESI-QqQ-MS/MS). In contrast to others [22-26] our method
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offers the simultaneous determination of hydroxy sterols and bile acids in one single run, in addition to
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the advantage of a quick and easy-to-use sample preparation and clean-up without further complex
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derivatization techniques. The chromatographic baseline separation of isomeric hydroxy sterols and
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bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. In this
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paper, the method is validated in 6 biological matrices and additionally, applicability is demonstrated
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in a mouse feeding study. The method achieved a high dynamic range in different biological matrices
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and was successfully applied for the determination of sterols in blood plasma, urine, bile, feces, adi-
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pose tissue and liver.
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2. Theory
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All sterols are based on the same steroid with a hydroxyl-group at the 3-position of the A-ring and
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often only differ in the structural position of mainly polar functional groups. As shown in Figure 1,
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this results in many isobaric compounds that cannot be determined quantitatively via MRM-MS-
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applications due to identical precursor- and fragment-masses without prior baseline separation [22].
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The steroid backbone is mainly of nonpolar character, thus liquid chromatography is performed using
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reversed phase conditions. On the one hand, this results in good retardation for nearly all steroid com-
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pounds but selectivity is often poor when it comes to isobaric structures only differing in the position-
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ing of a polar functional group (Figure 1). On the other hand, the application of mainly polar station-
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ary phases like hydrophilic interaction liquid chromatography- (HILIC) columns often shows elution
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with the solvent front or is characterized by poor selectivity. In order to separate isobaric sterols by
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liquid chromatography, an approach requires a stationary phase with hydrophobic properties such as a
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C18 reversed phase- (RP) that is combined with polar selectivity. In principle, there are two types of
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RP-based stationary phases with polar selectivity: polar endcapped and polar embedded phases. For
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endcapping, accessible peripheral silanol groups of the C18-RP are endcapped with polar groups. For
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polar embedding, the polar groups are located within the alkyl-chain. Figure 2 shows a schematic rep-
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resentation of the fundamental construction of a polar embedded stationary phase. Polar carbamate-,
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urea-, ether- and amide groups are commonly used to increase the polar selectivity that is mainly me-
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diated by hydrogen bonding interactions [27].
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3. Materials and methods
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3.1 Chemicals
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Methanol (LC-MS-grade) and ammonium acetate (≥ 97 %, p.a.) were purchased from Carl Roth
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(Karlsruhe, Germany). LC-MS grade water was obtained from Merck Millipore Ultra-pure water puri-
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fication system (Merck Millipore, Darmstadt, Germany). Formic acid (FA) was of 99+ % purity and
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obtained from Acros Organics (Geel, Belgium). The following bile acids: CDCA, UDCA, HDCA, α-
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MCA, β-MCA, ω-MCA, GLCA, GUDCA, TLCA, TUDCA, THDCA, T-α-MCA, T-β-MCA and d4-
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GCDCA and following steroid hormones: progesterone, pregnanolone, pregnanedione and THDOC
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were purchased from Steraloids, Inc. (Newport, RI, USA). All further standards including TCA, GCA,
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GCDCA, GDCA, TCDCA, DOC, CA, DCA, TDCA, stigmasterol, and dried bovine bile were pur-
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chased from Sigma-Aldrich Chemie GmbH (Munich, Germany). 7-OH-Chol, 22-S-OH-Chol, 24-S-
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OH-Chol, d6-25-OH-Chol, 27-OH-Chol-, 25-OH-Chol, d7-Chol, cholesterol and desmosterol were
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obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All abbreviations are shown in Table 1.
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3.2 Standard solutions and calibration
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Standard solutions of individual bile acids and steroid hormones were prepared by dissolving the re-
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spective compounds separately in methanol, whereas sterol standards including the deuterated internal
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standards d4-GCDCA, d6-25-OH-Chol, d7-Chol were prepared in Folch reagent [28]. All solutions
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were stored at -20 °C. A nine-point calibration curve was constructed. For every calibration point, the
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internal standard (IS) was used at a final concentration of 10 μM for d4-GCDCA, 10 μM for d6-25-
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OH-Chol and 100 μM for d7-Chol, respectively. For quantification of bile acids and steroid hormones,
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calculation of analyte levels was performed based on the internal standard d4-GCDCA. The sterols 7- ,
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22-S-, 24-S-, 25- and 27-OH-Chol were corrected for d6-25-OH-Chol whereas levels for cholesterol,
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desmosterol, stigmasterol, sitosterol are based on the internal standard d7-Chol.
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3.3 Sample preparation
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3.3.1 Solid phase extraction
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Solid phase extraction was performed according to the method of Lund and Diczfalusy [29]. To moni-
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tor the best recovery rates of representative analytes (cholic acid, cholesterol, stigmasterol), eleven
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different compositions of solvents (compare Suppl. Table 1) used for washing and elution were tested.
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3.3.2 Extraction
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EDTA-plasma and urine samples were obtained from healthy volunteers. Samples were stored at
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-20°C until analysis. 70 μL MeOH and 10 μL IS-mix containing 100 μM d4-GCDCA, 100 μM d6-25-
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OH-Chol and 1000 μM d7-Chol were added to 20 μL plasma. The samples were vortexed for three
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times, shaken continuously over a period of 10 minutes and centrifuged (12,000 g, 10 minutes). The
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supernatants were transferred into glass vials and analyzed by LC-MS/MS. Urine samples (20 µl)
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spiked with 10 µl of IS were processed similarly as plasma samples.
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Liver and adipose tissue: 150 mg of liver or adipose tissues obtained from fasted wild type C57BL/6J
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mice were spiked with 10 μL IS-solution and mixed with 3 mL methanol followed by homogenization
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with an Ultra-Turrax. After 10 minutes of centrifugation (12,000 g), supernatants were transferred to
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new glass vials and evaporated in vacuo until dryness. The dried lipid-containing samples were dis-
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solved in 100 μL methanol.
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Feces: Fecal samples obtained from mice were lyophilized and then dried samples were pestled using
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a mortar. 5 mg of the homogenic powder were extracted with 1000 μL MeOH containing IS-mixture
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(100 μmol/L d4-GCDCA, 100 μmol/L d6-25-OH-Chol, 1000 μmol/L d7-Chol). After sonication the
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samples were centrifuged and the supernatants were used for LC-MS/MS analysis.
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3.4 LC-MS-analysis
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Liquid chromatography was performed on a Accucore™ Polar Premium HPLC column (2.6 μm, 150
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mm x 2.1 mm i.d.), equipped with an Accucore™ Polar Premium defender guard column (Thermo
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Fischer Scientific Inc., Waltham, MA, USA), at 20°C with a flow rate of 300 μL/min using an Agilent
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1200 Infinity Quaternary LC System (Agilent Technologies, Waldbronn, Germany). The mobile phase
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A was water, and B was MeOH, both containing 0.1 % formic acid and ammonium acetate at 5 mM.
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The gradient elution started with 40 % A for 2 minutes, linearly increased to 100 % B in 18 minutes,
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which was kept constant for 20 minutes and brought back to 40 % A in 3 minutes followed by 13 min-
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utes of re-equilibration. For matrix abundant samples, step 3 was extended for another period of 10
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minutes. The injection volume for all samples was 5 μL. For detection a QqQ-MS/MS API 4000 Q
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trap (Applied Biosystems, Darmstadt, Germany) equipped with a turbo ion spray source, operating in
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positive ion mode was used with the following mass spectrometer settings: ion spray voltage = 4500
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V; ion source heater = 550°C; source gas 1 = 40 psi; source gas 2 = 50 psi and curtain gas = 20 psi.
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Analytes were identified by their chromatographic characteristics and their specific fragmentations,
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using mass transitions between precursor and product ions for each analyte (Table 1). The entire LC-
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MS/MS-system was controlled using Analyst 1.6.1 software (AB Sciex, Concord, Ontario, Canada).
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3.5 Method validation
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Validation of the method was performed either in absence (base calibration) or presence (matrix cali-
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bration) of the following matrices: plasma, bile, urine, liver, fat and feces. Linearity, recovery, lower
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limit of quantification (LLoQ), precision and accuracy were assessed in analogy to accepted guidelines
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[30].
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Linearity: Linearity was determined by analysis of calibration curves for all commercially available
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standards of bile acids/ hydroxy sterols. The method was validated using a nine-point calibration curve
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(n = 5). To asses matrix effects, fixed volumes of each matrix were spiked three times (n = 3) with
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different volumes of the appropriate mix-standard solution to construct a calibration curve of six cali-
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bration points. The concentration ranges for baseline calibration are shown in Table 2.
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Recovery: To evaluate the efficiency of the extraction procedure a known concentration of IS-mixture
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was added to all matrices before and after extraction (n = 3). The samples were treated as described in
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section 3.2. The integrated average peak area ratio of each analyte before extraction was compared to
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the corresponding peak area ratio in samples after extraction. The recovery rate (R) was determined as
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R (%) = concentrationbefore/concentrationafter x 100.
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Lower limit of quantification:
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The lower limit of quantification has been set as the lowest standard on the calibration curve as it met
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the following conditions: The analyte response was at least 5 times the response compared to a blank
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response and the analyte peak is identifiable, discrete, and reproducible.
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Precision and accuracy:
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Precision was obtained determining the coefficient of variation of every analyte of 15 samples on 3
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different concentration levels.
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Accuracy was obtained determining the concentration of every analyte of 15 samples on 3 different
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concentration levels. The maximal variance between the value determined and the true value is 15 %
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or 20 % at the LLOQ.
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3.6 Experimental animals and diets
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All animal experiments were approved by the Animal Welfare Officers of University Medical Center
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Hamburg-Eppendorf and Behörde für Gesundheit und Verbraucherschutz Hamburg. Wild type
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C57BL/6J mice were bred in the animal facility of Universal Medical Center Hamburg-Eppendorf
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with a day and night cycle of 12 hours with ad libitum access to food and water. Male age-matched
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(12 weeks) mice were housed at 22°-24°C in single cages and fed either a standard chow diet or a
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Western Type diet (WTD) containing 21 % total fat and 0.2 % cholesterol (ssniff EF R/M acc.
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TD88137 mod.) for one week. At day 6, cages were cleaned and feces were collected on day 7 after a
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24 hours period. Blood samples were collected in EDTA-coated tubes (Sarstedt, Nümbrecht, Deutsch-
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land) by cardiac puncture of anaesthetized mice after 4 hours of fasting on day 7. Then, organs were
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harvested after systemic perfusion with phosphate buffered saline (PBS, Life Technologies, Darm-
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stadt, Germany) via the left heart ventricle and immediately stored at -80°C.
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4. Results and discussion
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As bile acids and hydroxy sterols are important regulators of cellular metabolism and inflammatory
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processes [6-9] their simultaneous determination is of great interest. Up to our knowledge this is the
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first LC-MS-approach combining the determination of bile acids and hydroxy sterols.
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4.1
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Previously described methods predominantly apply GC to separate positional isomeric sterol com-
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pounds. While this may result in very good separation results, further sample preparation steps includ-
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ing complex derivatization techniques are needed [31,32]. The advantage of using polar-embedded-
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LC-MS-techniques for this analytical challenge is that baseline separation and detection of positional
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isomeric sterol compounds can be achieved without any preceding derivatization steps. To obtain
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MRM-transitions, 37 single compound solutions (0.1 mM in methanol enriched with 5 mM NH4Ac)
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were directly injected into the ESI source in positive ionization mode. NH4Ac supplementation
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thereby increased signal intensity of the analytes in positive ionization mode. Optimization of the dif-
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ferent MS-parameters was accomplished by means of the auto tuning mode provided by the Analyst
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1.6. software. Due to the use of NH4Ac as modifier, ammonia adducts were predominantly detected as
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most abundant precursor ions (Table 1). Fragmentation resulted in the release of either H2O (m/z 18),
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NH3 + H2O (m/z 35) or NH3 + 2 H2O (m/z 53) (Table 1, Figure 3). As described above, analyzed
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compounds share obvious structural similarities and are partially isobaric, and therefore fragmentize in
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a similar or even identical manner (Figure 3). Therefore, high-resolution separation is obligatory to
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identify single sterol derivatives. The substantial improvement of chromatographic separation of the
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embedded RP18 column in comparison to a conventional RP18 column is shown in Figure 2 a, b. No-
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tably, most prominent differences are observed in chromatographic resolution of isobaric muricholates
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as well as hydroxy sterols (Figure 2 a insets). In general, peak performance was enhanced strongly
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compared to the RP18 column. Best peak performance was achieved by adding 5 mM NH4Ac and 0.1
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% FA as modifiers to mobile phases (Figure 2 a-b). In order to combine demands of sensitivity and
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sufficient data acquisition rates the MRM method was split into three periods.
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4.2
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In contrast to methods employing solid phase extraction (SPE) as a classical procedure [22,26,29] we
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used a simpler liquid-liquid-methanol extraction protocol, as we observed small recovery rates for
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representative analytes such as cholesterol or cholic acid when performing previously described SPE
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procedures on normal-phase SPE cartridges (Suppl. Figure 1). Normal-phase SPE was used in order to
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reduce nonpolar compounds, such as triacylglycerols and cholesterol esters and, in addition, to apply a
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complementary sample cleanup prior to a reversed phase analytical method. The obtained data shows
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that a selective sample cleanup via SPE for both, bile acids and hydroxy sterols is not possible in a
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single SPE run. However, using a simple methanol extraction we obtained good recovery rates for
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representative analytes, even in the presence of complex matrices (Suppl. Table 2, Figure 5). For re-
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covery analyses, we added known concentrations of the internal standards to 6 different matrices either
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before or after extraction, performed as described in Section 3. For 94 % of the representative analytes
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and the different matrices the recovery was above 75 % (Suppl. Table 3).
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The obtained results indicate that simple and fast liquid-liquid-extraction protocols can be an efficient
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alternative to laborious SPE approaches.
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4.3
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Validation of the method was performed either in absence (base calibration) or presence (matrix cali-
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bration) of the following matrices: plasma, bile, urine, liver, adipose tissue, feces. As many published
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methods apply complex sample clean-ups such as SPE [22,26], it was important to show that the liq-
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uid-liquid extraction used here leads to reproducible, reasonable, and precise results.
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Method validation
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4.3.1
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97 % of all analytes showed acceptable linearity (R > 0.99) in a broad dynamic range of 10 - 500- fold
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(Table 2). Representative calibration curves and corresponding residual plots for UDCA and 7-OH-
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Cholesterol are shown in Figures 4 a and 4 b, respectively. According to the guidelines of the FDA the
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coefficient of variation (CV) was < 15 % for ~ ¾ of the analytes in base calibration. Depending on the
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respective analyte, the lower limit of quantification ranged between 9 nM and 2 µM (Table 2).
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4.3.2
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To overcome matrix effects, linearity responses were studied in blank matrices (urine, EDTA-plasma,
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feces, bile, fat and liver) spiked with mix-standard solution and IS-mixture. The correlation coeffi-
Baseline calibration
Matrix calibration
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cients of all the calibration curves in the different matrices were at least 0.999 for 82 % of the analytes
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(see Suppl. Table 3 a-f). CV was between 2.5 % and 15 % for ⅔ of the analytes, which demonstrated a
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minor influence of matrix effects on our method. The calibration data involving calibration curve
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equations, curve parameters, and R2 values are presented for each matrix in Suppl. Table 3 a-f.
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4.3.3
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The obtained values for precision and accuracy (Table 3) at the LLOQ were below 20 % for 90 % of
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the analytes. The values for expected concentrations were below 15 % for 90 % the analytes. Thus
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almost all values met the conditions stated by the FDA [30].
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4.4
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As bile acids and hydroxy sterols have recently gained growing interest [4,15,18,33], our method to
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rapidly quantify cholesterol intermediates of the de novo synthesis and excretion pathways provides a
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powerful tool to depict and trace the metabolic state of different tissues and to trail enterohepatic cir-
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culation. As described before [15,33] diet influences the bile acid pool in mice. Therefore, body fluids,
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feces and organs of wild type C57BL/6J mice fed either a normal chow or a WTD were processed to
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quantify sterols and its bile acid derivatives. Our observed concentrations and composition of bile
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acids and hydroxy sterols (Figure 5) are in line with previous studies using GC-MS- or LC-MS-based
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methods [7,16,17,24,25,33,34]. We did observe that concentrations for bile acids in feces were quite
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variable. One reason for this is the strong impact of the diverse microbiome on fecal bile acid content
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and composition [16,35,36]. As shown in Figure 5 (log scale) and Suppl. Figure 2 (linear scale), the
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diet influences the concentration of specific hydroxylated sterols and bile acids in liver, adipose tissue,
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feces, and plasma (Figure 5a-d) but not in gall bladder (Suppl. Figure 3). The most prominent change
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was a decreased concentration of stigmasterol in feces of WTD-fed mice (Figure 5 c). The difference
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in stigmasterol can be explained by the composition of the diet as the plant based chow diet contains a
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higher content of phytosterols compared to the milk fat-based WTD. Although the WTD is enriched in
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cholesterol, we could only detect a significant increase of cholesterol in WAT after WTD feeding
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(Figure 5 b). As expected, WAT contained the lowest concentrations of bile acids in comparison to the
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other analyzed organs (Figure 5 a-d). Notably, even in tissues with a low content of bile acids such as
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the adipose tissue, most bile acid species could be detected. The broad spectrum of matrices applicable
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to this method results in a comprehensive picture of systemic bile acid and hydroxy sterol metabolism.
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This opens the perspective to delineate the functional roles of sterols and bile acids in the regulation of
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metabolism via G-protein coupled receptors and ligand-activated transcription factors. Moreover this
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method might have the potential to be implemented in clinical screening approaches and patient moni-
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toring in the future.
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5
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Here, we describe a simple, effective and sensitive LC-MS/MS-based protocol to quantify 34 sterols
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and bile acids using a polar embedded stationary phase. It is highly applicable to different biological
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compartments such as blood, urine, feces, bile, liver and adipose tissue. The simultaneous quantifica-
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Method application
Conclusion
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tion of bile acids, sterols and even isobaric hydroxylated cholesterol derivatives provides a powerful
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tool for the investigation of cholesterol utilizing metabolic pathways in response to environmental or
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genetic alterations.
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[7]
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[16]
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[3]
K. Bloch, The biological synthesis of cholesterol, Science 150 (1965) 19-28. A.A. Kandutsch, A.E. Russell, Preputial gland tumor sterols. 3. A metabolic pathway from lanosterol to cholesterol, Journal of Biological Chemistry 235 (1960) 2256-2261. F.R. Maxfield, I. Tabas, Role of cholesterol and lipid organization in disease, Nature 438 (2005) 612-621. F. Kuipers, V.W. Bloks, A.K. Groen, Beyond intestinal soap-bile acids in metabolic control, Nat Rev Endocrinol (2014). D.W. Russell, The enzymes, regulation, and genetics of bile acid synthesis, Annu Rev Biochem 72 (2003) 137-174. A.C. Calkin, P. Tontonoz, Transcriptional integration of metabolism by the nuclear sterolactivated receptors LXR and FXR, Nat Rev Mol Cell Biol 13 (2012) 213-224. M. Umetani, P. Ghosh, T. Ishikawa, J. Umetani, M. Ahmed, C. Mineo, P.W. Shaul, The Cholesterol Metabolite 27-Hydroxycholesterol Promotes Atherosclerosis via Proinflammatory Processes Mediated by Estrogen Receptor Alpha, Cell Metab 20 (2014) 172182. A. Reboldi, E.V. Dang, J.G. McDonald, L. Guosheng, D.W. Russell, J.G. Cyster, 25Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon, Science 345 (2014) 679-684. E.R. Nelson, S.E. Wardell, J.S. Jasper, S. Park, S. Suchindran, M.K. Howe, N.J. Carver, R.V. Pillai, P.M. Sullivan, V. Sondhi, M. Umetani, J. Geradts, D.P. McDonnell, 27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology, Science 342 (2013) 1094-1098. W. Song, W.M. Pierce, Jr., R.A. Prough, R.N. Redinger, Characteristics of cholesterol 7 alphahydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of rodent liver, Biochem Pharmacol 41 (1991) 1439-1447. N.B. Javitt, Biologic role(s) of the 25(R),26-hydroxycholesterol metabolic pathway, Biochim Biophys Acta 1529 (2000) 136-141. E.G. Lund, J.M. Guileyardo, D.W. Russell, cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain, Proc Natl Acad Sci U S A 96 (1999) 72387243. E. Lund, I. Bjorkhem, C. Furster, K. Wikvall, 24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver, Biochim Biophys Acta 1166 (1993) 177182. J. Li-Hawkins, E.G. Lund, A.D. Bronson, D.W. Russell, Expression cloning of an oxysterol 7 alpha-hydroxylase selective for 24-hydroxycholesterol, Journal of Biological Chemistry 275 (2000) 16543-16549. M. Watanabe, S.M. Houten, C. Mataki, M.A. Christoffolete, B.W. Kim, H. Sato, N. Messaddeq, J.W. Harney, O. Ezaki, T. Kodama, K. Schoonjans, A.C. Bianco, J. Auwerx, Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation, Nature 439 (2006) 484-489. S.I. Sayin, A. Wahlstrom, J. Felin, S. Jantti, H.U. Marschall, K. Bamberg, B. Angelin, T. Hyotylainen, M. Oresic, F. Backhed, Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist, Cell Metab 17 (2013) 225-235. F. Li, C. Jiang, K.W. Krausz, Y. Li, I. Albert, H. Hao, K.M. Fabre, J.B. Mitchell, A.D. Patterson, F.J. Gonzalez, Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity, Nat Commun 4 (2013) 2384.
pt
[1] [2]
Ac ce
299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344
Page 9 of 21
[24] [25]
[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
ip t
cr
[23]
us
[22]
an
[21]
M
[20]
ed
[19]
K. Ryan, V. Tremaroli, C. Clemmensen, P. Kovatcheva-Datchary, A. Myronovych, R. Karns, H. Wilson-Pérez, D. Sandoval, R. Kohli, F. Bäckhed, R. Seeley, FXR is a molecular target for the effects of vertical sleeve gastrectomy, Nature 509 (2014) 183-188. J. Li-Hawkins, M. Gafvels, M. Olin, E.G. Lund, U. Andersson, G. Schuster, I. Bjorkhem, D.W. Russell, G. Eggertsen, Cholic acid mediates negative feedback regulation of bile acid synthesis in mice, Journal of Clinical Investigation 110 (2002) 1191-1200. X. Hu, Y. Bonde, G. Eggertsen, M. Rudling, Muricholic bile acids are potent regulators of bile acid synthesis via a positive feedback mechanism, J Intern Med 275 (2014) 27-38. D.Q. Wang, S. Tazuma, D.E. Cohen, M.C. Carey, Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse, Am J Physiol Gastrointest Liver Physiol 285 (2003) G494-502. J.G. McDonald, B.M. Thompson, E.C. McCrum, D.W. Russell, Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry, Methods Enzymol 432 (2007) 145-170. J. Acimovic, A. Lovgren-Sandblom, K. Monostory, D. Rozman, M. Golicnik, D. Lutjohann, I. Bjorkhem, Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells, J Chromatogr B Analyt Technol Biomed Life Sci 877 (2009) 2081-2086. J.C. Garcia-Canaveras, M.T. Donato, C.J. V., A. Lahoz, Targted profiling of circulating and hepatic bile acids in human, mouse, and rat usimng a UPLC-MRM-MS-validated method, Journal of Lipid Research 53 (2012). S.J. Tsai, Y.S. Zhong, J.F. Weng, H.H. Huang, P.Y. Hsieh, Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms, J Chromatogr A 1218 (2011) 524-533. G. Kakiyama, A. Muto, H. Takei, H. Nittono, T. Murai, T. Kurosawa, A.F. Hofmann, W.M. Pandak, J.S. Bajaj, A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS, J Lipid Res 55 (2014) 978-990. J.W. Coym, Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases, J Sep Sci 31 (2008) 1712-1718. J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipides from animal tissues, Journal of Biological Chemistry 226 (1957) 497-509. E.G. Lund, U. Diczfalusy, Quantitation of receptor ligands by mass spectrometry, Methods Enzymol 364 (2003) 24-37. FDA, Guidance for Industry - Bioanalytical Method Validation, U.S. Departement of Health and Human Services (2001) Center for Drug Evaluation and Research, Center for Veterinary medicine. J.J. Birk, M. Dippold, G.L. Wiesenberg, B. Glaser, Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatographymass spectrometry, J Chromatogr A 1242 (2012) 1-10. S. Matysik, G. Schmitz, Application of gas chromatography-triple quadrupole mass spectrometry to the determination of sterol components in biological samples in consideration of the ionization mode, Biochimie 95 (2013) 489-495. Y. Qi, C. Jiang, J. Cheng, K.W. Krausz, T. Li, J.M. Ferrell, F.J. Gonzalez, J.Y. Chiang, Bile acid signaling in lipid metabolism: Metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice, Biochim Biophys Acta (2014). M. Umetani, H. Domoto, A.K. Gormley, I.S. Yuhanna, C.L. Cummins, N.B. Javitt, K.S. Korach, P.W. Shaul, D.J. Mangelsdorf, 27-hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen, Nature Medicine 13 (2007) 1185-1192. L.A. David, C.F. Maurice, R.N. Carmody, D.B. Gootenberg, J.E. Button, B.E. Wolfe, A.V. Ling, A.S. Devlin, Y. Varma, M.A. Fischbach, S.B. Biddinger, R.J. Dutton, P.J. Turnbaugh, Diet rapidly and reproducibly alters the human gut microbiome, Nature 505 (2014) 559-563. P.J. Turnbaugh, Microbiology: fat, bile and gut microbes, Nature 487 (2012) 47-48.
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398 ACKNOWLEDGEMENT
400
The authors thank Meike Kröger for excellent technical assistance. J.H. is supported by a grant from
401
the DFG (SFB 841: Liver inflammation: infection, immune regulation and consequences) and by EU
402
FP7 project RESOLVE (FP7-HEALTH-2012-305707).
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Figure 1 Structural isomerism of cholesterol-derived compounds; (a) Cholesterol, (b) (I) CA, (II) α-MCA, (III) ω-MCA, (IV) β-MCA, (c) (I) 22-OH-Chol (II) 27-OH-Chol, (III) 25-OH-Chol, (IV) 24-OH-Chol Figure 2 Total ion chromatograms (TIC) of a standard mixture containing 34 bile acids and oxidized sterol derivatives separated on a (a) polar embedded C18 column and (b) on a conventional C18 column. The polar embedded stationary phase (scheme a) is equipped with multiple polar groups which are either located within the C18 chain or on remaining free silica groups. The conventional C18 column contains only aliphatic alkyl chains (scheme b). Baseline separation of isobaric compounds such as bile acids (e.g. ω-MCA, α-MCA, β-MCA, and CA) as well as hydroxy sterols (e.g. 22-OH-Chol, 24-OHChol, 25-OH-Chol, 27-OH-Chol) is only achieved using the polar embedded column (compare (XICs) a and b). Figure 3 Electrospray ionization product ion mass spectra of nominated (a) bile acids and (b) sterols. A (I) αMCA, (II) β-MCA, (III) ω-MCA, (IV) CA; B (I) 22-S-OH-Chol, (II) 24-S-OH-Chol, (III) 25-OHChol, (IV) 27-OH-Chol Figure 4 Exemplary calibration curves and corresponding residual plots for (a) UDCA (base calibration), (b) 7OH-Cholesterol (base calibration), (c) UDCA (matrix calibration, feces) and (d) 7-OH-Cholesterol (matrix calibration, feces) Figure 5 Concentration of different oxidized cholesterol derivatives and bile acids in (a) Liver, (b) adipose tissue (AT), (c) feces, (d) plasma of wild type mice fed either a chow diet (open bars) or WTD (filled bars) for one week (data are expressed in mean +/- SEM, n = 5, ***P < 0.001, **P < 0.01, *P < 0.05).
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*Highlights (for review)
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1. Simultaneous quantification of 34 hydroxy sterols and bile acids via LC-ESI-MS/MS. 2. Rapid and simple sample clean-up without complex derivatization techniques or solid phase extraction. 3. Validated for six different biological matrices: plasma, liver, adipose tissue, urine, gall bladder and feces. 4. Chromatographic baseline separation of multiple isobaric compounds using a novel polar embedded stationary phase. 5. Good method application was illustrated taking the example of a mice feeding study.
Page 12 of 21
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Tables Table 1 Operating parameters in MRM-mode
C27H46O2
402.65
M + AA
C27H46O2
402.65
M + AA
C27H46O2
402.65
M-H2O
C24H40O4
392.57
M + H+
C27H46O
386.67
M + AA
Cholic acid (CA)
C24H40O5
408.57
M + AA
Deoxycholic acid (DCA) Desmosterol
C24H40O4
392.57
M + AA
C27H44O
384.64
M + AA
Desoxycorticosterone (DOC) Glycochenodeoxycholic acid (GCDCA) Glycocholic acid (GCA) Glycodeoxycholic acid (GDCA) Glycolithocholic acid (GLCA) Glycoursodeoxycholic acid (GUDCA) Hyodeoxycholic acid (HDCA) Pregnanedione
C21H30O3
330.46
M + H+
C26H43NO5
449.62
M + AA
C26H43NO6
465.62
M + AA
pt
ce
Ac C26H43NO5
449.62
M + AA
C26H43NO4
433.62
M + AA
C26H43NO5
449.62
M + AA
C24H40O4
392.57
M + AA
C21H32O2
316.48
M + H+
111 111 111 106 106 106 106 106 106 111 111 111 91 91 91 76 76 76 101 101 101 51 51 51 1 1 1 101 101 101 101 101 101 36 36 36 41 41 41 41 41 41 36 36 36 36 36 36 76 76 76 91 91 91
CE (V) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
CXP (V) 19 61 11 17 53 11 9 13 15 33 63 13 19 31 79 5 63 11 37 59 11 27 99 19 13 65 21 57 51 11 33 73 39 25 21 13 29 25 15 27 19 13 21 51 13 23 17 11 9 9 15 19 75 15
10 14 12 10 12 12 12 12 10 12 6 12 10 12 14 12 14 10 12 12 10 10 14 10 10 12 12 14 6 10 16 12 18 12 14 12 12 12 14 12 12 14 12 12 14 12 12 14 12 12 10 8 14 8
ip t
M + AA
EP (V)
cr
402.65
DP (V)
us
C27H46O2
22-S-Hydroxycholesterol (22-OH-Chol) 24-S-Hydroxycholesterol (24-OH-Chol) 25-Hydroxycholesterol (25-OH-Chol) 27-Hydroxycholesterol (27-OH-Chol) 7-Hydroxycholesterol (7-OH-Chol) Chenodeoxycholic acid (CDCA) Cholesterol (Chol)
Fragments (m/z) Q1 420.44 → 367.3 Q2 420.44 → 81.1 Qnt 420.44 → 385.3 Q1 420.39 → 367.3 Q2 420.39 → 69.1 Qnt 420.39 → 385.3 Q1 420.35 → 402.3 Q2 420.35 → 385.3 Qnt 420.35 → 367.3 Q1 420.38 → 161.1 Q2 420.38 → 81.1 Qnt 420.38 → 385.3 Q1 385.32 → 367.2 Q2 385.32 → 159.1 Qnt 385.32 → 91.0 Q1 393.30 → 375.2 Q2 393.30 → 81.1 Qnt 393.30 → 357.3 Q1 404.41 → 147.2 Q2 404.41 → 81.1 Qnt 404.41 → 369.3 Q1 426.34 → 355.2 Q2 426.34 → 91.0 Qnt 426.34 → 373.2 Q1 410.32 → 375.2 Q2 410.32 → 81.1 Qnt 410.32 → 67.0 Q1 402.40 → 81.0 Q2 402.40 → 95.1 Qnt 402.40 → 367.3 Q1 331.20 → 109.0 Q2 331.20 → 97.0 Qnt 331.20 → 79.0 Q1 467.41 → 414.2 Q2 467.41 → 432.2 Qnt 467.41 → 450.2 Q1 483.36 → 412.3 Q2 483.36 → 430.2 Qnt 483.36 → 466.2 Q1 467.39 → 414.3 Q2 467.39 → 432.3 Qnt 467.39 → 450.3 Q1 451.39 → 416.3 Q2 451.39 → 76.0 Qnt 451.39 → 434.3 Q1 467.40 → 414.2 Q2 467.40 → 432.2 Qnt 467.40 → 450.2 Q1 410.24 → 393.2 Q2 410.24 → 375.2 Qnt 410.24 → 357.2 Q1 317.24 → 299.2 Q2 317.24 → 281.2 Qnt 317.24 → 91.0
an
402.65
M+X+ (m/z) M + AA
M
M (g/mol)
ed
Molecular formula C27H46O2
Compound
Q1 = qualifier 1, Q2 = qualifier 2, Qnt = quantifier, M + AA = ammonia - adduct, M + H+ = proton - adduct, DP = declustering potential, Page EP = entrance potential, CE = collision energy, CXP = collision cell exit potential
18 of 21
Table 1 Operating parameters in MRM-mode
C21H30O2
314.46
M + H+
Stigmasterol
C29H48O
412.69
M + AA
M + AA
Q2 Qnt Q1
430.41 → 55.0 430.41 → 395.3 517.33 → 500.3
M + AA
Q2 Qnt Q1
517.33 → 482.2 517.33 → 464.3 533.33 → 462.2
M + AA
Q2 Qnt Q1
M + AA
Q2 Qnt Q1
M + AA
Q2 Qnt Q1
C26H45NO6S
Taurohyodeoxycholic acid (THDCA)
C26H45NO6S
Taurolithocholic acid (TLCA)
C26H45NO5S
Tauroursodeoxycholic acid (TUDCA)
C26H45NO6S
Tauro-αmuricholic acid (T-α-MCA)
C26H45NO7S
Tauro-βmuricholic acid (T-β-MCA)
C26H45NO7S
Tetrahydrodesoxycorticosterone (THDOC) Ursodeoxycholic acid (UDCA)
C21H34O4
ω-Muricholic acid (ω-MCA)
499.70
483.71
499.70
515.70
515.70
334.49
Ac
α-Muricholic acid (α-MCA) β-Muricholic acid (β-MCA)
499.70
71 71 76
10 10 10
19 27 31
14 14 14
533.33 → 498.2 533.33 → 516.2 517.35 → 464.3
76 76 71
10 10 10
21 17 29
14 16 14
517.35 → 482.2 517.35 → 500.2 517.32 → 464.2
71 71 71
10 10 10
23 15 23
14 16 14
517.32 → 482.3 517.32 → 500.2 501.35 → 484.2
71 71 76
10 10 10
19 13 13
14 14 14
M + AA
Q2 Qnt Q1
501.35 → 126.0 501.35 → 466.2 517.32 → 500.2
76 76 76
10 10 10
49 23 13
8 14 16
M + 2*AA
Q2 Qnt Q1
517.32 → 482.2 517.32 → 464.2 550.26 → 221.0
76 76 46
10 10 10
17 25 27
14 14 18
M + AA
Q2 Qnt Q1
550.26 → 73.0 550.26 → 533.3 533.25 → 516.2
46 46 76
10 10 10
89 9 15
12 16 16
Q2 Qnt Q1
533.25 → 498.2 533.25 → 480.3 352.27 → 317.1
76 76 41
10 10 10
19 25 19
14 14 8
Q2 Qnt
352.27 → 299.1 352.27 → 335.2
41 41
10 10
21 13
8 10
C24H40O4
392.57
Q1
410.18 → 81.0
21
10
65
12
Q2 Qnt Q1 Q2 Qnt Q1
410.18 → 91.1 410.18 → 357.2 426.31 → 355.3 426.31 → 391.3 426.31 → 373.3 426.29 → 373.2
21 21 41 41 41 56
10 10 10 10 10 10
99 19 25 13 17 19
14 10 10 12 10 10
Q2 Qnt Q1 Q2 Qnt
426.29 → 355.2 426.29 → 391.2 426.30 → 355.2 426.30 → 391.2 426.30 → 373.2
56 56 41 41 41
10 10 10 10 10
25 15 23 13 17
10 12 10 12 10
M + AA
M + AA
C24H40O5
408.57
M + AA
C24H40O5
408.57
M + AA
C24H40O5
408.57
M + AA
us
Taurodeoxycholic acid (TDCA)
515.70
8 10 8 18 18 16 6 8 12 16
an
C26H45NO7S
23 11 19 37 37 33 35 77 11 13
ed
Taurocholic acid (TCA)
499.70
CXP (V)
10 10 10
pt
C26H45NO6S
46 46 46 41 41 41 126
EP (V) CE (V) 10 10 10 10 10 10 10
126 126 71
ce
Taurochenodeoxycholic acid (TCDCA)
DP (V)
ip t
Progesterone
Fragments (m/z) Q1 336.34 → 283.2 Q2 336.34 → 319.2 Qnt 336.34 → 301.2 Q1 316.28 → 109.1 Q2 316.28 → 315.2 Qnt 316.28 → 97.0 Q1 430.41 → 83.1
cr
318.49
M+X+ (m/z) M + AA
M (g/mol)
M
Pregnanolone
Molecular formula C21H34O2
Compound
Q1 = qualifier 1, Q2 = qualifier 2, Qnt = quantifier, M + AA = ammonia - adduct, M + H+ = proton - adduct, DP = declustering potential, EP = entrance potential, CE = collision energy, CXP = collision cell exit potential
Page 19 of 21
22-S-OH-Chol 24-S-/25-OH-Chol 27-OH-Chol 7-OH-Chol CDCA/DCA Chol CA Desmosterol DOC GCDCA GCA GDCA GLCA GUDCA HDCA Pregnanedione Pregnanolone Progesterone Stigmasterol TCDCA TCA TDCA THDCA TLCA TUDCA T-α/β-MCA THDOC UDCA α-MCA β-MCA ω-MCA
0.999 0.997 0.996 0.999 0.999 0.991 0.999 0.958 0.999 0.998 0.999 0.999 0.999 0.998 0.994 0.995 0.998 0.999 0.997 0.999 0.999 0.999 0.999 0.998 0.998 0.999 0.996 0.996 0.999 0.999 0.999
y = 3,417x - 645 y = 1,888x - 80 y = 6,193x - 1,180 y = 964,882x - 27,753 y = 49,161x - 5,374 y = 3,831x + 2,253 y = 1E+06x - 56,934 y = 1,971x + 4,652 y = 1E+07x + 1E+06 y = 602,142x - 62,201 y = 1E+06x - 25,293 y =685,171x + 45,121 y = 515,926x - 3,676 y = 466,848x - 16,405 y = 12,264x + 6,158 y = 2.97E+06x + 79,433 y = 923,334x - 11,104 y = 3E06x + 15,618 y = 587x + 4,535 y = 1E+06x - 42,278 y = 1E+06x - 29,816 y = 803,720x - 21,728 y = 386,660x - 5,493 y = 3E+06x - 10,327 y = 694,933x - 20,462 y = 802,232x - 4,972 y = 2E+06x + 24,953 y = 124,576x + 3,094 y = 1E+06x - 19,493 y = 2E+06x - 970 y = 646,768x + 12,653
0.300 0.300 0.200 0.100 0.360 0.600 0.180 1.000 0.180 0.180 0.060 0.180 0.018 0.060 0.300 0.180 0.030 0.018 2.000 0.060 0.060 0.060 0.030 0.018 0.060 0.036 0.060 0.180 0.030 0.009 0.060
ed
pt
1,455 1,149 1,687 139,694 11,172 3,049 116,475 2,502 1,692,780 66,166 68,529 86,557 61,955 54,745 7,244 374,076 134,904 39,710 708 155,666 112,036 54,846 31,955 307,453 80,598 122,764 224,181 20,241 192,283 89,104 61,145
SD (µM)
CV (%) 0.43 0.61 0.27 0.14 0.23 0.80 0.11 1.27 0.11 0.11 0.06 0.13 0.12 0.12 0.59 0.13 0.15 0.01 1.21 0.12 0.11 0.07 0.08 0.12 0.12 0.15 0.12 0.16 0.14 0.05 0.09
21.7 20.5 28.7 13.0 9.7 20.3 9.0 26.8 9.6 9.3 6.3 10.7 16.0 11.8 11.9 10.7 17.1 10.8 30.8 12.6 10.9 6.9 9.7 16.1 11.7 10.2 11.9 13.8 16.1 13.1 9.4
Ac
ce
RSD (counts)
cr
LLoQ (µM)
us
Regression equation
an
R
M
Analytes
ip t
Table 2 Calibration curves and figures of merit of the baseline calibration
LLoQ = lower limit of quantification (µM) , RSD = residual standard deviation (counts), SD = standard deviation for the procedure (µM), CV = coefficient of variation (%)
Page 20 of 21
Table 3 Accuracy and precision for three concentration levels
Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ip t
GCDCA C (µM) (%) 0,20 20,00 0,60 4,70 3,00 2,70 0,20 18,20 0,60 12,80 3,00 2,50
CA C (µM) (%) 0,20 19,20 0,60 17,10 3,00 3,30 0,20 3,70 0,60 3,90 3,00 0,40
Desmosterol C (µM) (%) 3,00 15,20 6,00 20,80 10,00 13,20 3,00 12,30 6,00 7,10 10,00 9,90
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
GCA C (µM) (%) 0,10 19,70 0,60 6,90 3,00 3,50 0,10 13,70 0,60 6,00 3,00 3,50
GDCA C (µM) (%) 0,20 19,40 0,60 10,50 3,00 6,60 0,20 16,80 0,60 6,10 3,00 5,90
GLCA C (µM) (%) 0,02 4,70 0,30 9,70 3,00 10,40 0,02 16,30 0,30 9,70 3,00 10,50
GUDCA C (µM) (%) 0,10 21,40 0,60 6,40 3,00 7,00 0,10 18,10 0,60 3,60 3,00 6,20
HDCA C (µM) (%) 0,90 12,50 9,00 6,80 15,00 4,20 0,90 20,00 3,00 6,10 15,00 4,20
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
Pregnanedione C (µM) (%) 0,20 18,40 0,60 11,60 3,00 3,50 0,20 11,90 0,60 13,00 3,00 2,80
Pregnanolone C (µM) (%) 0,03 17,20 0,60 8,60 3,00 8,00 0,03 18,90 0,60 11,50 3,00 8,50
Progesterone C (µM) (%) 0,20 11,90 0,60 7,80 3,00 5,80 0,20 11,50 0,60 13,40 3,00 5,60
Stigmasterol C (µM) (%) 2,00 16,10 6,00 6,70 10,00 7,20 2,00 55,60 6,00 15,90 10,00 15,50
TCDCA C (µM) (%) 0,10 16,10 0,60 6,70 3,00 7,20 0,10 16,70 0,60 8,00 3,00 8,10
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
TCA C (µM) (%) 0,10 19,00 0,60 7,30 3,00 5,90 0,10 17,70 0,60 6,30 3,00 5,80
TDCA C (µM) (%) 0,10 19,90 0,60 8,70 3,00 3,50 0,10 15,20 0,60 7,20 3,00 3,60
THDCA C (µM) (%) 0,03 16,10 0,60 6,40 3,00 3,70 0,03 19,00 0,60 5,30 3,00 3,70
TLCA C (µM) (%) 0,02 18,40 0,30 12,50 3,00 9,10 0,02 17,70 0,30 7,10 3,00 9,30
TUDCA C (µM) (%) 0,10 18,30 0,60 12,30 3,00 2,90 0,10 11,00 0,60 17,30 3,00 2,60
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
T-α/β-MCA C (µM) (%) 0,04 9,30 0,60 5,50 6,00 5,10 0,04 7,50 0,60 2,10 6,00 0,80
THDOC C (µM) (%) 0,10 22,00 0,60 19,00 3,00 3,60 0,10 17,10 0,60 17,30 3,00 3,10
UDCA C (µM) (%) 0,20 9,40 1,80 7,90 0,30 7,90 0,20 17,00 1,80 3,70 0,30 4,80
α-MCA C (µM) (%) 0,03 11,10 0,60 8,90 3,00 8,30 0,03 14,20 0,60 6,40 3,00 8,30
β-MCA C (µM) (%) 0,01 17,50 0,20 9,50 1,50 7,00 0,01 13,80 0,20 4,90 1,50 7,20
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
ω-MCA C (µM) (%) 0,10 18,70 C = concentration, ACC = accuracy, Prec = precision 0,30 6,30 3,00 5,10 0,10 17,90 0,30 5,30 3,00 5,20
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C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
(%) 6,00 16,20 12,40 17,00 10,40 11,40
M
ACC (%)
DOC C (µM) (%) 0,20 11,00 0,60 4,80 3,00 5,00 0,20 14,30 0,60 4,40 3,00 5,00
Chol C (µM) 0,60 2,00 10,00 0,60 2,00 10,00
ed
Prec (%)
CDCA/DCA C (µM) (%) 0,20 19,80 0,60 3,70 3,00 4,40 0,20 14,30 0,60 1,10 3,00 5,10
pt
ACC (%)
7-OH-Chol C (µM) (%) 0,10 16,90 1,00 8,50 5,00 5,10 0,10 15,60 1,00 13,30 5,00 5,00
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Prec (%)
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
27-OH-Chol C (µM) (%) 0,20 19,00 1,00 19,20 2,00 23,30 0,20 39,80 1,00 23,00 2,00 28,80
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ACC (%)
22-S-OH-Chol 24-S-/25-OH-Chol C (µM) (%) C (µM) (%) 0,30 10,10 0,60 18,00 3,00 12,00 3,80 12,20 5,00 13,50 6,30 11,00 0,30 29,10 0,60 25,20 3,00 16,20 3,80 11,90 5,00 14,10 6,30 11,10
Page 21 of 21
S0021-9673(14)01655-0 http://dx.doi.org/doi:10.1016/j.chroma.2014.10.064 CHROMA 355942
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
5-9-2014 20-10-2014 21-10-2014
Please cite this article as: C. John, P. Werner, A. Worthmann, K. Wegner, K. T¨odter, L. Scheja, S. Rohn, J. Heeren, M. Fischer, A liquid chromatographytandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids, Journal of Chromatography A (2014), http://dx.doi.org/10.1016/j.chroma.2014.10.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A liquid chromatography-tandem mass spectrometry-based method for the simultaneous de-
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termination of hydroxy sterols and bile acids
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Clara John1*, Philipp Werner2*, Anna Worthmann1, Katrin Wegner2, Klaus Tödter1, Ludger Scheja1,
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Sascha Rohn2, Joerg Heeren1# and Markus Fischer2#
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* These authors contributed equally
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1
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Eppendorf, Martinistr. 52, 20246 Hamburg; 2Hamburg School of Food Science, Institute of Food
Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-
Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany
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#
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Markus Fischer, Hamburg School of Food Science, Institute of Food Chemistry, University of Ham-
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burg, Grindelallee 117, 20146 Hamburg, Germany. Phone: +49(0)40-42838-4342, Email: mar-
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[email protected]
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Joerg Heeren, University Medical Center Hamburg-Eppendorf, Dept. of Biochemistry and Molecular
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Cell Biology, Martinistraße 52, 20246 Hamburg, Germany. Phone: +49(0)40-4710-54745, Email:
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[email protected]
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Corresponding Authors:
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Abstract
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Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators
23
of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid
24
species requires powerful analytical tools to quantify these structurally and chemically similar ana-
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lytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based
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method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as
27
their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols
28
and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase.
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The current method features a simple extraction protocol validated for blood plasma, urine, gall blad-
30
der, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization proce-
31
dures. The total extraction recovery for representative analytes ranged between 58-86 % in plasma, 85
32
% in urine, 79-92 % in liver, 76-98 % in adipose tissue, 93-104 % in feces and 62-79 % in gall blad-
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der. The validation procedure demonstrated that the calibration curves were linear over the selected
34
concentration ranges for 97 % of the analytes, with calculated coefficients of determination (R2) of
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greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched
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Western type diet illustrated that the protocol described here provides a powerful tool to simultane-
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ously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the
38
enterohepatic circulation.
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1. Introduction
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Sterols are apolar lipids formed by four fused rings resulting in an inflexible core. While plants syn-
41
thesize diverse phytosterols, cholesterol is the most abundant sterol synthesized in animal cells. Its
42
chemical structure allows cholesterol to serve as a component of membranes, facilitating membrane
43
fluidity, as well as a precursor of steroid hormones and other signaling molecules. In carnivores, cho-
44
lesterol supply is provided either by diet or by de novo synthesis [1,2].
45
High intracellular cholesterol levels are cytotoxic and a disturbed cholesterol metabolism observed in
46
patients with hypercholesterolemia is causally associated with the development of cardiovascular dis-
47
ease [3]. An important way to get rid of excess cholesterol is the biliary excretion of either non-
48
esterified cholesterol or its hydrophilic derivatives, the bile acids (BA) [4,5]Besides their role as in-
49
termediates in the BA synthesis pathway, hydroxy sterols are well known agonists for the transcription
50
factor liver X receptor and the estrogen receptor α (ERα), thereby regulating systemic lipid and energy
51
metabolism [6] as well as inflammatory processes known to be important for the development and
52
progression of chronic metabolic diseases such as cardiovascular disease, type 2 diabetes mellitus and
53
cancer [7-9]. In a multi-step reaction catalyzed by enzymes located in the liver [5,10-14], hydroxy
54
sterols are converted into primary bile acids (PBA). In humans, cholic acid (CA) and chenodeoxy-
55
cholic acid (CDCA) are the main primary bile acids whereas CA and muricholic acid (MCA) pre-
56
dominate in mice [5]. Further conjugation to taurine (predominant in rodents) or glycine (predominant
57
in humans) increases hydrophilicity. Via bile ducts BA reach the intestine where they facilitate absorp-
58
tion of dietary fats. Secondary bile acids (SBA) arise from action of the gut microbiota [4]. Besides
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their function in fat absorption, BA have signaling functions, acting as agonists and antagonists of the
60
transcription factor farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (GBAR1
61
also known as TGR5). These receptors mediate bile acid, lipid, and glucose homeostasis as well as
62
energy expenditure [6,15-18]. The multifunctional presence of hydroxy sterols and bile acids in differ-
63
ent biochemical pathways shows that their quantitative observation in different biofluids and organs is
64
of great scientific relevance, especially with respect to the study of chronic inflammatory metabolic
65
diseases. Unfortunately, there are very few analytical methods for the determination of hydroxy sterols
66
and bile acids in one analytical approach. A bottleneck to the analysis of hydroxy sterols and bile acids
67
is the chromatographic separation prior to analysis via mass-spectrometric detection. As many of the
68
analytes are isobaric and very similar in their structure, no selective multiple reaction monitoring-
69
(MRM) transitions can be obtained. This results in the need for chromatographic baseline separation.
70
In the past, this problem has often been solved by determining very similar compounds as a sum pa-
71
rameter or via gas chromatography (GC)-MS-approaches [19]. GC-based applications are able to sepa-
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rate positional isomeric sterol compounds but need further sample preparation steps including complex
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derivatization techniques. As sterols and their derivatives can have very different biochemical func-
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tions, these procedures are not purposeful for some scientific research questions [20,21].
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In this work, our aim was to provide a method for the quantitative determination of 34 sterols in bio-
76
logical matrices using high performance liquid chromatography electrospray ionization triple quadru-
77
pol tandem mass spectrometry (HPLC-ESI-QqQ-MS/MS). In contrast to others [22-26] our method
78
offers the simultaneous determination of hydroxy sterols and bile acids in one single run, in addition to
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the advantage of a quick and easy-to-use sample preparation and clean-up without further complex
80
derivatization techniques. The chromatographic baseline separation of isomeric hydroxy sterols and
81
bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. In this
82
paper, the method is validated in 6 biological matrices and additionally, applicability is demonstrated
83
in a mouse feeding study. The method achieved a high dynamic range in different biological matrices
84
and was successfully applied for the determination of sterols in blood plasma, urine, bile, feces, adi-
85
pose tissue and liver.
86
2. Theory
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All sterols are based on the same steroid with a hydroxyl-group at the 3-position of the A-ring and
88
often only differ in the structural position of mainly polar functional groups. As shown in Figure 1,
89
this results in many isobaric compounds that cannot be determined quantitatively via MRM-MS-
90
applications due to identical precursor- and fragment-masses without prior baseline separation [22].
91
The steroid backbone is mainly of nonpolar character, thus liquid chromatography is performed using
92
reversed phase conditions. On the one hand, this results in good retardation for nearly all steroid com-
93
pounds but selectivity is often poor when it comes to isobaric structures only differing in the position-
94
ing of a polar functional group (Figure 1). On the other hand, the application of mainly polar station-
95
ary phases like hydrophilic interaction liquid chromatography- (HILIC) columns often shows elution
96
with the solvent front or is characterized by poor selectivity. In order to separate isobaric sterols by
97
liquid chromatography, an approach requires a stationary phase with hydrophobic properties such as a
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C18 reversed phase- (RP) that is combined with polar selectivity. In principle, there are two types of
99
RP-based stationary phases with polar selectivity: polar endcapped and polar embedded phases. For
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endcapping, accessible peripheral silanol groups of the C18-RP are endcapped with polar groups. For
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polar embedding, the polar groups are located within the alkyl-chain. Figure 2 shows a schematic rep-
102
resentation of the fundamental construction of a polar embedded stationary phase. Polar carbamate-,
103
urea-, ether- and amide groups are commonly used to increase the polar selectivity that is mainly me-
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diated by hydrogen bonding interactions [27].
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3. Materials and methods
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3.1 Chemicals
107
Methanol (LC-MS-grade) and ammonium acetate (≥ 97 %, p.a.) were purchased from Carl Roth
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(Karlsruhe, Germany). LC-MS grade water was obtained from Merck Millipore Ultra-pure water puri-
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fication system (Merck Millipore, Darmstadt, Germany). Formic acid (FA) was of 99+ % purity and
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obtained from Acros Organics (Geel, Belgium). The following bile acids: CDCA, UDCA, HDCA, α-
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MCA, β-MCA, ω-MCA, GLCA, GUDCA, TLCA, TUDCA, THDCA, T-α-MCA, T-β-MCA and d4-
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GCDCA and following steroid hormones: progesterone, pregnanolone, pregnanedione and THDOC
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were purchased from Steraloids, Inc. (Newport, RI, USA). All further standards including TCA, GCA,
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GCDCA, GDCA, TCDCA, DOC, CA, DCA, TDCA, stigmasterol, and dried bovine bile were pur-
115
chased from Sigma-Aldrich Chemie GmbH (Munich, Germany). 7-OH-Chol, 22-S-OH-Chol, 24-S-
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OH-Chol, d6-25-OH-Chol, 27-OH-Chol-, 25-OH-Chol, d7-Chol, cholesterol and desmosterol were
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obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All abbreviations are shown in Table 1.
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3.2 Standard solutions and calibration
119
Standard solutions of individual bile acids and steroid hormones were prepared by dissolving the re-
120
spective compounds separately in methanol, whereas sterol standards including the deuterated internal
121
standards d4-GCDCA, d6-25-OH-Chol, d7-Chol were prepared in Folch reagent [28]. All solutions
122
were stored at -20 °C. A nine-point calibration curve was constructed. For every calibration point, the
123
internal standard (IS) was used at a final concentration of 10 μM for d4-GCDCA, 10 μM for d6-25-
124
OH-Chol and 100 μM for d7-Chol, respectively. For quantification of bile acids and steroid hormones,
125
calculation of analyte levels was performed based on the internal standard d4-GCDCA. The sterols 7- ,
126
22-S-, 24-S-, 25- and 27-OH-Chol were corrected for d6-25-OH-Chol whereas levels for cholesterol,
127
desmosterol, stigmasterol, sitosterol are based on the internal standard d7-Chol.
128
3.3 Sample preparation
129
3.3.1 Solid phase extraction
130
Solid phase extraction was performed according to the method of Lund and Diczfalusy [29]. To moni-
131
tor the best recovery rates of representative analytes (cholic acid, cholesterol, stigmasterol), eleven
132
different compositions of solvents (compare Suppl. Table 1) used for washing and elution were tested.
133
3.3.2 Extraction
134
EDTA-plasma and urine samples were obtained from healthy volunteers. Samples were stored at
135
-20°C until analysis. 70 μL MeOH and 10 μL IS-mix containing 100 μM d4-GCDCA, 100 μM d6-25-
136
OH-Chol and 1000 μM d7-Chol were added to 20 μL plasma. The samples were vortexed for three
137
times, shaken continuously over a period of 10 minutes and centrifuged (12,000 g, 10 minutes). The
138
supernatants were transferred into glass vials and analyzed by LC-MS/MS. Urine samples (20 µl)
139
spiked with 10 µl of IS were processed similarly as plasma samples.
140
Liver and adipose tissue: 150 mg of liver or adipose tissues obtained from fasted wild type C57BL/6J
141
mice were spiked with 10 μL IS-solution and mixed with 3 mL methanol followed by homogenization
142
with an Ultra-Turrax. After 10 minutes of centrifugation (12,000 g), supernatants were transferred to
143
new glass vials and evaporated in vacuo until dryness. The dried lipid-containing samples were dis-
144
solved in 100 μL methanol.
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Feces: Fecal samples obtained from mice were lyophilized and then dried samples were pestled using
146
a mortar. 5 mg of the homogenic powder were extracted with 1000 μL MeOH containing IS-mixture
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(100 μmol/L d4-GCDCA, 100 μmol/L d6-25-OH-Chol, 1000 μmol/L d7-Chol). After sonication the
148
samples were centrifuged and the supernatants were used for LC-MS/MS analysis.
149
3.4 LC-MS-analysis
150
Liquid chromatography was performed on a Accucore™ Polar Premium HPLC column (2.6 μm, 150
151
mm x 2.1 mm i.d.), equipped with an Accucore™ Polar Premium defender guard column (Thermo
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Fischer Scientific Inc., Waltham, MA, USA), at 20°C with a flow rate of 300 μL/min using an Agilent
153
1200 Infinity Quaternary LC System (Agilent Technologies, Waldbronn, Germany). The mobile phase
154
A was water, and B was MeOH, both containing 0.1 % formic acid and ammonium acetate at 5 mM.
155
The gradient elution started with 40 % A for 2 minutes, linearly increased to 100 % B in 18 minutes,
156
which was kept constant for 20 minutes and brought back to 40 % A in 3 minutes followed by 13 min-
157
utes of re-equilibration. For matrix abundant samples, step 3 was extended for another period of 10
158
minutes. The injection volume for all samples was 5 μL. For detection a QqQ-MS/MS API 4000 Q
159
trap (Applied Biosystems, Darmstadt, Germany) equipped with a turbo ion spray source, operating in
160
positive ion mode was used with the following mass spectrometer settings: ion spray voltage = 4500
161
V; ion source heater = 550°C; source gas 1 = 40 psi; source gas 2 = 50 psi and curtain gas = 20 psi.
162
Analytes were identified by their chromatographic characteristics and their specific fragmentations,
163
using mass transitions between precursor and product ions for each analyte (Table 1). The entire LC-
164
MS/MS-system was controlled using Analyst 1.6.1 software (AB Sciex, Concord, Ontario, Canada).
165
3.5 Method validation
166
Validation of the method was performed either in absence (base calibration) or presence (matrix cali-
167
bration) of the following matrices: plasma, bile, urine, liver, fat and feces. Linearity, recovery, lower
168
limit of quantification (LLoQ), precision and accuracy were assessed in analogy to accepted guidelines
169
[30].
170
Linearity: Linearity was determined by analysis of calibration curves for all commercially available
171
standards of bile acids/ hydroxy sterols. The method was validated using a nine-point calibration curve
172
(n = 5). To asses matrix effects, fixed volumes of each matrix were spiked three times (n = 3) with
173
different volumes of the appropriate mix-standard solution to construct a calibration curve of six cali-
174
bration points. The concentration ranges for baseline calibration are shown in Table 2.
175
Recovery: To evaluate the efficiency of the extraction procedure a known concentration of IS-mixture
176
was added to all matrices before and after extraction (n = 3). The samples were treated as described in
177
section 3.2. The integrated average peak area ratio of each analyte before extraction was compared to
178
the corresponding peak area ratio in samples after extraction. The recovery rate (R) was determined as
179
R (%) = concentrationbefore/concentrationafter x 100.
180
Lower limit of quantification:
181
The lower limit of quantification has been set as the lowest standard on the calibration curve as it met
182
the following conditions: The analyte response was at least 5 times the response compared to a blank
183
response and the analyte peak is identifiable, discrete, and reproducible.
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Precision and accuracy:
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Precision was obtained determining the coefficient of variation of every analyte of 15 samples on 3
186
different concentration levels.
187
Accuracy was obtained determining the concentration of every analyte of 15 samples on 3 different
188
concentration levels. The maximal variance between the value determined and the true value is 15 %
189
or 20 % at the LLOQ.
190
3.6 Experimental animals and diets
191
All animal experiments were approved by the Animal Welfare Officers of University Medical Center
192
Hamburg-Eppendorf and Behörde für Gesundheit und Verbraucherschutz Hamburg. Wild type
193
C57BL/6J mice were bred in the animal facility of Universal Medical Center Hamburg-Eppendorf
194
with a day and night cycle of 12 hours with ad libitum access to food and water. Male age-matched
195
(12 weeks) mice were housed at 22°-24°C in single cages and fed either a standard chow diet or a
196
Western Type diet (WTD) containing 21 % total fat and 0.2 % cholesterol (ssniff EF R/M acc.
197
TD88137 mod.) for one week. At day 6, cages were cleaned and feces were collected on day 7 after a
198
24 hours period. Blood samples were collected in EDTA-coated tubes (Sarstedt, Nümbrecht, Deutsch-
199
land) by cardiac puncture of anaesthetized mice after 4 hours of fasting on day 7. Then, organs were
200
harvested after systemic perfusion with phosphate buffered saline (PBS, Life Technologies, Darm-
201
stadt, Germany) via the left heart ventricle and immediately stored at -80°C.
202
4. Results and discussion
203
As bile acids and hydroxy sterols are important regulators of cellular metabolism and inflammatory
204
processes [6-9] their simultaneous determination is of great interest. Up to our knowledge this is the
205
first LC-MS-approach combining the determination of bile acids and hydroxy sterols.
206
4.1
207
Previously described methods predominantly apply GC to separate positional isomeric sterol com-
208
pounds. While this may result in very good separation results, further sample preparation steps includ-
209
ing complex derivatization techniques are needed [31,32]. The advantage of using polar-embedded-
210
LC-MS-techniques for this analytical challenge is that baseline separation and detection of positional
211
isomeric sterol compounds can be achieved without any preceding derivatization steps. To obtain
212
MRM-transitions, 37 single compound solutions (0.1 mM in methanol enriched with 5 mM NH4Ac)
213
were directly injected into the ESI source in positive ionization mode. NH4Ac supplementation
214
thereby increased signal intensity of the analytes in positive ionization mode. Optimization of the dif-
215
ferent MS-parameters was accomplished by means of the auto tuning mode provided by the Analyst
216
1.6. software. Due to the use of NH4Ac as modifier, ammonia adducts were predominantly detected as
217
most abundant precursor ions (Table 1). Fragmentation resulted in the release of either H2O (m/z 18),
218
NH3 + H2O (m/z 35) or NH3 + 2 H2O (m/z 53) (Table 1, Figure 3). As described above, analyzed
219
compounds share obvious structural similarities and are partially isobaric, and therefore fragmentize in
220
a similar or even identical manner (Figure 3). Therefore, high-resolution separation is obligatory to
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identify single sterol derivatives. The substantial improvement of chromatographic separation of the
222
embedded RP18 column in comparison to a conventional RP18 column is shown in Figure 2 a, b. No-
223
tably, most prominent differences are observed in chromatographic resolution of isobaric muricholates
224
as well as hydroxy sterols (Figure 2 a insets). In general, peak performance was enhanced strongly
225
compared to the RP18 column. Best peak performance was achieved by adding 5 mM NH4Ac and 0.1
226
% FA as modifiers to mobile phases (Figure 2 a-b). In order to combine demands of sensitivity and
227
sufficient data acquisition rates the MRM method was split into three periods.
228
4.2
229
In contrast to methods employing solid phase extraction (SPE) as a classical procedure [22,26,29] we
230
used a simpler liquid-liquid-methanol extraction protocol, as we observed small recovery rates for
231
representative analytes such as cholesterol or cholic acid when performing previously described SPE
232
procedures on normal-phase SPE cartridges (Suppl. Figure 1). Normal-phase SPE was used in order to
233
reduce nonpolar compounds, such as triacylglycerols and cholesterol esters and, in addition, to apply a
234
complementary sample cleanup prior to a reversed phase analytical method. The obtained data shows
235
that a selective sample cleanup via SPE for both, bile acids and hydroxy sterols is not possible in a
236
single SPE run. However, using a simple methanol extraction we obtained good recovery rates for
237
representative analytes, even in the presence of complex matrices (Suppl. Table 2, Figure 5). For re-
238
covery analyses, we added known concentrations of the internal standards to 6 different matrices either
239
before or after extraction, performed as described in Section 3. For 94 % of the representative analytes
240
and the different matrices the recovery was above 75 % (Suppl. Table 3).
241
The obtained results indicate that simple and fast liquid-liquid-extraction protocols can be an efficient
242
alternative to laborious SPE approaches.
243
4.3
244
Validation of the method was performed either in absence (base calibration) or presence (matrix cali-
245
bration) of the following matrices: plasma, bile, urine, liver, adipose tissue, feces. As many published
246
methods apply complex sample clean-ups such as SPE [22,26], it was important to show that the liq-
247
uid-liquid extraction used here leads to reproducible, reasonable, and precise results.
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Method validation
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4.3.1
250
97 % of all analytes showed acceptable linearity (R > 0.99) in a broad dynamic range of 10 - 500- fold
251
(Table 2). Representative calibration curves and corresponding residual plots for UDCA and 7-OH-
252
Cholesterol are shown in Figures 4 a and 4 b, respectively. According to the guidelines of the FDA the
253
coefficient of variation (CV) was < 15 % for ~ ¾ of the analytes in base calibration. Depending on the
254
respective analyte, the lower limit of quantification ranged between 9 nM and 2 µM (Table 2).
255
4.3.2
256
To overcome matrix effects, linearity responses were studied in blank matrices (urine, EDTA-plasma,
257
feces, bile, fat and liver) spiked with mix-standard solution and IS-mixture. The correlation coeffi-
Baseline calibration
Matrix calibration
Page 7 of 21
258
cients of all the calibration curves in the different matrices were at least 0.999 for 82 % of the analytes
259
(see Suppl. Table 3 a-f). CV was between 2.5 % and 15 % for ⅔ of the analytes, which demonstrated a
260
minor influence of matrix effects on our method. The calibration data involving calibration curve
261
equations, curve parameters, and R2 values are presented for each matrix in Suppl. Table 3 a-f.
262
4.3.3
263
The obtained values for precision and accuracy (Table 3) at the LLOQ were below 20 % for 90 % of
264
the analytes. The values for expected concentrations were below 15 % for 90 % the analytes. Thus
265
almost all values met the conditions stated by the FDA [30].
266
4.4
267
As bile acids and hydroxy sterols have recently gained growing interest [4,15,18,33], our method to
268
rapidly quantify cholesterol intermediates of the de novo synthesis and excretion pathways provides a
269
powerful tool to depict and trace the metabolic state of different tissues and to trail enterohepatic cir-
270
culation. As described before [15,33] diet influences the bile acid pool in mice. Therefore, body fluids,
271
feces and organs of wild type C57BL/6J mice fed either a normal chow or a WTD were processed to
272
quantify sterols and its bile acid derivatives. Our observed concentrations and composition of bile
273
acids and hydroxy sterols (Figure 5) are in line with previous studies using GC-MS- or LC-MS-based
274
methods [7,16,17,24,25,33,34]. We did observe that concentrations for bile acids in feces were quite
275
variable. One reason for this is the strong impact of the diverse microbiome on fecal bile acid content
276
and composition [16,35,36]. As shown in Figure 5 (log scale) and Suppl. Figure 2 (linear scale), the
277
diet influences the concentration of specific hydroxylated sterols and bile acids in liver, adipose tissue,
278
feces, and plasma (Figure 5a-d) but not in gall bladder (Suppl. Figure 3). The most prominent change
279
was a decreased concentration of stigmasterol in feces of WTD-fed mice (Figure 5 c). The difference
280
in stigmasterol can be explained by the composition of the diet as the plant based chow diet contains a
281
higher content of phytosterols compared to the milk fat-based WTD. Although the WTD is enriched in
282
cholesterol, we could only detect a significant increase of cholesterol in WAT after WTD feeding
283
(Figure 5 b). As expected, WAT contained the lowest concentrations of bile acids in comparison to the
284
other analyzed organs (Figure 5 a-d). Notably, even in tissues with a low content of bile acids such as
285
the adipose tissue, most bile acid species could be detected. The broad spectrum of matrices applicable
286
to this method results in a comprehensive picture of systemic bile acid and hydroxy sterol metabolism.
287
This opens the perspective to delineate the functional roles of sterols and bile acids in the regulation of
288
metabolism via G-protein coupled receptors and ligand-activated transcription factors. Moreover this
289
method might have the potential to be implemented in clinical screening approaches and patient moni-
290
toring in the future.
291
5
292
Here, we describe a simple, effective and sensitive LC-MS/MS-based protocol to quantify 34 sterols
293
and bile acids using a polar embedded stationary phase. It is highly applicable to different biological
294
compartments such as blood, urine, feces, bile, liver and adipose tissue. The simultaneous quantifica-
ip t
Precision and accuracy
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Method application
Conclusion
Page 8 of 21
295
tion of bile acids, sterols and even isobaric hydroxylated cholesterol derivatives provides a powerful
296
tool for the investigation of cholesterol utilizing metabolic pathways in response to environmental or
297
genetic alterations.
298
[7]
[8] [9] [10] [11] [12] [13] [14] [15]
[16]
[17]
ip t
cr
us
[6]
an
[5]
M
[4]
ed
[3]
K. Bloch, The biological synthesis of cholesterol, Science 150 (1965) 19-28. A.A. Kandutsch, A.E. Russell, Preputial gland tumor sterols. 3. A metabolic pathway from lanosterol to cholesterol, Journal of Biological Chemistry 235 (1960) 2256-2261. F.R. Maxfield, I. Tabas, Role of cholesterol and lipid organization in disease, Nature 438 (2005) 612-621. F. Kuipers, V.W. Bloks, A.K. Groen, Beyond intestinal soap-bile acids in metabolic control, Nat Rev Endocrinol (2014). D.W. Russell, The enzymes, regulation, and genetics of bile acid synthesis, Annu Rev Biochem 72 (2003) 137-174. A.C. Calkin, P. Tontonoz, Transcriptional integration of metabolism by the nuclear sterolactivated receptors LXR and FXR, Nat Rev Mol Cell Biol 13 (2012) 213-224. M. Umetani, P. Ghosh, T. Ishikawa, J. Umetani, M. Ahmed, C. Mineo, P.W. Shaul, The Cholesterol Metabolite 27-Hydroxycholesterol Promotes Atherosclerosis via Proinflammatory Processes Mediated by Estrogen Receptor Alpha, Cell Metab 20 (2014) 172182. A. Reboldi, E.V. Dang, J.G. McDonald, L. Guosheng, D.W. Russell, J.G. Cyster, 25Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon, Science 345 (2014) 679-684. E.R. Nelson, S.E. Wardell, J.S. Jasper, S. Park, S. Suchindran, M.K. Howe, N.J. Carver, R.V. Pillai, P.M. Sullivan, V. Sondhi, M. Umetani, J. Geradts, D.P. McDonnell, 27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology, Science 342 (2013) 1094-1098. W. Song, W.M. Pierce, Jr., R.A. Prough, R.N. Redinger, Characteristics of cholesterol 7 alphahydroxylase and 7 alpha-hydroxycholesterol hydroxylase activities of rodent liver, Biochem Pharmacol 41 (1991) 1439-1447. N.B. Javitt, Biologic role(s) of the 25(R),26-hydroxycholesterol metabolic pathway, Biochim Biophys Acta 1529 (2000) 136-141. E.G. Lund, J.M. Guileyardo, D.W. Russell, cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain, Proc Natl Acad Sci U S A 96 (1999) 72387243. E. Lund, I. Bjorkhem, C. Furster, K. Wikvall, 24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver, Biochim Biophys Acta 1166 (1993) 177182. J. Li-Hawkins, E.G. Lund, A.D. Bronson, D.W. Russell, Expression cloning of an oxysterol 7 alpha-hydroxylase selective for 24-hydroxycholesterol, Journal of Biological Chemistry 275 (2000) 16543-16549. M. Watanabe, S.M. Houten, C. Mataki, M.A. Christoffolete, B.W. Kim, H. Sato, N. Messaddeq, J.W. Harney, O. Ezaki, T. Kodama, K. Schoonjans, A.C. Bianco, J. Auwerx, Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation, Nature 439 (2006) 484-489. S.I. Sayin, A. Wahlstrom, J. Felin, S. Jantti, H.U. Marschall, K. Bamberg, B. Angelin, T. Hyotylainen, M. Oresic, F. Backhed, Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist, Cell Metab 17 (2013) 225-235. F. Li, C. Jiang, K.W. Krausz, Y. Li, I. Albert, H. Hao, K.M. Fabre, J.B. Mitchell, A.D. Patterson, F.J. Gonzalez, Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity, Nat Commun 4 (2013) 2384.
pt
[1] [2]
Ac ce
299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344
Page 9 of 21
[24] [25]
[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]
ip t
cr
[23]
us
[22]
an
[21]
M
[20]
ed
[19]
K. Ryan, V. Tremaroli, C. Clemmensen, P. Kovatcheva-Datchary, A. Myronovych, R. Karns, H. Wilson-Pérez, D. Sandoval, R. Kohli, F. Bäckhed, R. Seeley, FXR is a molecular target for the effects of vertical sleeve gastrectomy, Nature 509 (2014) 183-188. J. Li-Hawkins, M. Gafvels, M. Olin, E.G. Lund, U. Andersson, G. Schuster, I. Bjorkhem, D.W. Russell, G. Eggertsen, Cholic acid mediates negative feedback regulation of bile acid synthesis in mice, Journal of Clinical Investigation 110 (2002) 1191-1200. X. Hu, Y. Bonde, G. Eggertsen, M. Rudling, Muricholic bile acids are potent regulators of bile acid synthesis via a positive feedback mechanism, J Intern Med 275 (2014) 27-38. D.Q. Wang, S. Tazuma, D.E. Cohen, M.C. Carey, Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse, Am J Physiol Gastrointest Liver Physiol 285 (2003) G494-502. J.G. McDonald, B.M. Thompson, E.C. McCrum, D.W. Russell, Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry, Methods Enzymol 432 (2007) 145-170. J. Acimovic, A. Lovgren-Sandblom, K. Monostory, D. Rozman, M. Golicnik, D. Lutjohann, I. Bjorkhem, Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells, J Chromatogr B Analyt Technol Biomed Life Sci 877 (2009) 2081-2086. J.C. Garcia-Canaveras, M.T. Donato, C.J. V., A. Lahoz, Targted profiling of circulating and hepatic bile acids in human, mouse, and rat usimng a UPLC-MRM-MS-validated method, Journal of Lipid Research 53 (2012). S.J. Tsai, Y.S. Zhong, J.F. Weng, H.H. Huang, P.Y. Hsieh, Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms, J Chromatogr A 1218 (2011) 524-533. G. Kakiyama, A. Muto, H. Takei, H. Nittono, T. Murai, T. Kurosawa, A.F. Hofmann, W.M. Pandak, J.S. Bajaj, A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS, J Lipid Res 55 (2014) 978-990. J.W. Coym, Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases, J Sep Sci 31 (2008) 1712-1718. J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and purification of total lipides from animal tissues, Journal of Biological Chemistry 226 (1957) 497-509. E.G. Lund, U. Diczfalusy, Quantitation of receptor ligands by mass spectrometry, Methods Enzymol 364 (2003) 24-37. FDA, Guidance for Industry - Bioanalytical Method Validation, U.S. Departement of Health and Human Services (2001) Center for Drug Evaluation and Research, Center for Veterinary medicine. J.J. Birk, M. Dippold, G.L. Wiesenberg, B. Glaser, Combined quantification of faecal sterols, stanols, stanones and bile acids in soils and terrestrial sediments by gas chromatographymass spectrometry, J Chromatogr A 1242 (2012) 1-10. S. Matysik, G. Schmitz, Application of gas chromatography-triple quadrupole mass spectrometry to the determination of sterol components in biological samples in consideration of the ionization mode, Biochimie 95 (2013) 489-495. Y. Qi, C. Jiang, J. Cheng, K.W. Krausz, T. Li, J.M. Ferrell, F.J. Gonzalez, J.Y. Chiang, Bile acid signaling in lipid metabolism: Metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice, Biochim Biophys Acta (2014). M. Umetani, H. Domoto, A.K. Gormley, I.S. Yuhanna, C.L. Cummins, N.B. Javitt, K.S. Korach, P.W. Shaul, D.J. Mangelsdorf, 27-hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen, Nature Medicine 13 (2007) 1185-1192. L.A. David, C.F. Maurice, R.N. Carmody, D.B. Gootenberg, J.E. Button, B.E. Wolfe, A.V. Ling, A.S. Devlin, Y. Varma, M.A. Fischbach, S.B. Biddinger, R.J. Dutton, P.J. Turnbaugh, Diet rapidly and reproducibly alters the human gut microbiome, Nature 505 (2014) 559-563. P.J. Turnbaugh, Microbiology: fat, bile and gut microbes, Nature 487 (2012) 47-48.
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398 ACKNOWLEDGEMENT
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The authors thank Meike Kröger for excellent technical assistance. J.H. is supported by a grant from
401
the DFG (SFB 841: Liver inflammation: infection, immune regulation and consequences) and by EU
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FP7 project RESOLVE (FP7-HEALTH-2012-305707).
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Figure 1 Structural isomerism of cholesterol-derived compounds; (a) Cholesterol, (b) (I) CA, (II) α-MCA, (III) ω-MCA, (IV) β-MCA, (c) (I) 22-OH-Chol (II) 27-OH-Chol, (III) 25-OH-Chol, (IV) 24-OH-Chol Figure 2 Total ion chromatograms (TIC) of a standard mixture containing 34 bile acids and oxidized sterol derivatives separated on a (a) polar embedded C18 column and (b) on a conventional C18 column. The polar embedded stationary phase (scheme a) is equipped with multiple polar groups which are either located within the C18 chain or on remaining free silica groups. The conventional C18 column contains only aliphatic alkyl chains (scheme b). Baseline separation of isobaric compounds such as bile acids (e.g. ω-MCA, α-MCA, β-MCA, and CA) as well as hydroxy sterols (e.g. 22-OH-Chol, 24-OHChol, 25-OH-Chol, 27-OH-Chol) is only achieved using the polar embedded column (compare (XICs) a and b). Figure 3 Electrospray ionization product ion mass spectra of nominated (a) bile acids and (b) sterols. A (I) αMCA, (II) β-MCA, (III) ω-MCA, (IV) CA; B (I) 22-S-OH-Chol, (II) 24-S-OH-Chol, (III) 25-OHChol, (IV) 27-OH-Chol Figure 4 Exemplary calibration curves and corresponding residual plots for (a) UDCA (base calibration), (b) 7OH-Cholesterol (base calibration), (c) UDCA (matrix calibration, feces) and (d) 7-OH-Cholesterol (matrix calibration, feces) Figure 5 Concentration of different oxidized cholesterol derivatives and bile acids in (a) Liver, (b) adipose tissue (AT), (c) feces, (d) plasma of wild type mice fed either a chow diet (open bars) or WTD (filled bars) for one week (data are expressed in mean +/- SEM, n = 5, ***P < 0.001, **P < 0.01, *P < 0.05).
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*Highlights (for review)
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1. Simultaneous quantification of 34 hydroxy sterols and bile acids via LC-ESI-MS/MS. 2. Rapid and simple sample clean-up without complex derivatization techniques or solid phase extraction. 3. Validated for six different biological matrices: plasma, liver, adipose tissue, urine, gall bladder and feces. 4. Chromatographic baseline separation of multiple isobaric compounds using a novel polar embedded stationary phase. 5. Good method application was illustrated taking the example of a mice feeding study.
Page 12 of 21
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Figure 5
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Tables Table 1 Operating parameters in MRM-mode
C27H46O2
402.65
M + AA
C27H46O2
402.65
M + AA
C27H46O2
402.65
M-H2O
C24H40O4
392.57
M + H+
C27H46O
386.67
M + AA
Cholic acid (CA)
C24H40O5
408.57
M + AA
Deoxycholic acid (DCA) Desmosterol
C24H40O4
392.57
M + AA
C27H44O
384.64
M + AA
Desoxycorticosterone (DOC) Glycochenodeoxycholic acid (GCDCA) Glycocholic acid (GCA) Glycodeoxycholic acid (GDCA) Glycolithocholic acid (GLCA) Glycoursodeoxycholic acid (GUDCA) Hyodeoxycholic acid (HDCA) Pregnanedione
C21H30O3
330.46
M + H+
C26H43NO5
449.62
M + AA
C26H43NO6
465.62
M + AA
pt
ce
Ac C26H43NO5
449.62
M + AA
C26H43NO4
433.62
M + AA
C26H43NO5
449.62
M + AA
C24H40O4
392.57
M + AA
C21H32O2
316.48
M + H+
111 111 111 106 106 106 106 106 106 111 111 111 91 91 91 76 76 76 101 101 101 51 51 51 1 1 1 101 101 101 101 101 101 36 36 36 41 41 41 41 41 41 36 36 36 36 36 36 76 76 76 91 91 91
CE (V) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
CXP (V) 19 61 11 17 53 11 9 13 15 33 63 13 19 31 79 5 63 11 37 59 11 27 99 19 13 65 21 57 51 11 33 73 39 25 21 13 29 25 15 27 19 13 21 51 13 23 17 11 9 9 15 19 75 15
10 14 12 10 12 12 12 12 10 12 6 12 10 12 14 12 14 10 12 12 10 10 14 10 10 12 12 14 6 10 16 12 18 12 14 12 12 12 14 12 12 14 12 12 14 12 12 14 12 12 10 8 14 8
ip t
M + AA
EP (V)
cr
402.65
DP (V)
us
C27H46O2
22-S-Hydroxycholesterol (22-OH-Chol) 24-S-Hydroxycholesterol (24-OH-Chol) 25-Hydroxycholesterol (25-OH-Chol) 27-Hydroxycholesterol (27-OH-Chol) 7-Hydroxycholesterol (7-OH-Chol) Chenodeoxycholic acid (CDCA) Cholesterol (Chol)
Fragments (m/z) Q1 420.44 → 367.3 Q2 420.44 → 81.1 Qnt 420.44 → 385.3 Q1 420.39 → 367.3 Q2 420.39 → 69.1 Qnt 420.39 → 385.3 Q1 420.35 → 402.3 Q2 420.35 → 385.3 Qnt 420.35 → 367.3 Q1 420.38 → 161.1 Q2 420.38 → 81.1 Qnt 420.38 → 385.3 Q1 385.32 → 367.2 Q2 385.32 → 159.1 Qnt 385.32 → 91.0 Q1 393.30 → 375.2 Q2 393.30 → 81.1 Qnt 393.30 → 357.3 Q1 404.41 → 147.2 Q2 404.41 → 81.1 Qnt 404.41 → 369.3 Q1 426.34 → 355.2 Q2 426.34 → 91.0 Qnt 426.34 → 373.2 Q1 410.32 → 375.2 Q2 410.32 → 81.1 Qnt 410.32 → 67.0 Q1 402.40 → 81.0 Q2 402.40 → 95.1 Qnt 402.40 → 367.3 Q1 331.20 → 109.0 Q2 331.20 → 97.0 Qnt 331.20 → 79.0 Q1 467.41 → 414.2 Q2 467.41 → 432.2 Qnt 467.41 → 450.2 Q1 483.36 → 412.3 Q2 483.36 → 430.2 Qnt 483.36 → 466.2 Q1 467.39 → 414.3 Q2 467.39 → 432.3 Qnt 467.39 → 450.3 Q1 451.39 → 416.3 Q2 451.39 → 76.0 Qnt 451.39 → 434.3 Q1 467.40 → 414.2 Q2 467.40 → 432.2 Qnt 467.40 → 450.2 Q1 410.24 → 393.2 Q2 410.24 → 375.2 Qnt 410.24 → 357.2 Q1 317.24 → 299.2 Q2 317.24 → 281.2 Qnt 317.24 → 91.0
an
402.65
M+X+ (m/z) M + AA
M
M (g/mol)
ed
Molecular formula C27H46O2
Compound
Q1 = qualifier 1, Q2 = qualifier 2, Qnt = quantifier, M + AA = ammonia - adduct, M + H+ = proton - adduct, DP = declustering potential, Page EP = entrance potential, CE = collision energy, CXP = collision cell exit potential
18 of 21
Table 1 Operating parameters in MRM-mode
C21H30O2
314.46
M + H+
Stigmasterol
C29H48O
412.69
M + AA
M + AA
Q2 Qnt Q1
430.41 → 55.0 430.41 → 395.3 517.33 → 500.3
M + AA
Q2 Qnt Q1
517.33 → 482.2 517.33 → 464.3 533.33 → 462.2
M + AA
Q2 Qnt Q1
M + AA
Q2 Qnt Q1
M + AA
Q2 Qnt Q1
C26H45NO6S
Taurohyodeoxycholic acid (THDCA)
C26H45NO6S
Taurolithocholic acid (TLCA)
C26H45NO5S
Tauroursodeoxycholic acid (TUDCA)
C26H45NO6S
Tauro-αmuricholic acid (T-α-MCA)
C26H45NO7S
Tauro-βmuricholic acid (T-β-MCA)
C26H45NO7S
Tetrahydrodesoxycorticosterone (THDOC) Ursodeoxycholic acid (UDCA)
C21H34O4
ω-Muricholic acid (ω-MCA)
499.70
483.71
499.70
515.70
515.70
334.49
Ac
α-Muricholic acid (α-MCA) β-Muricholic acid (β-MCA)
499.70
71 71 76
10 10 10
19 27 31
14 14 14
533.33 → 498.2 533.33 → 516.2 517.35 → 464.3
76 76 71
10 10 10
21 17 29
14 16 14
517.35 → 482.2 517.35 → 500.2 517.32 → 464.2
71 71 71
10 10 10
23 15 23
14 16 14
517.32 → 482.3 517.32 → 500.2 501.35 → 484.2
71 71 76
10 10 10
19 13 13
14 14 14
M + AA
Q2 Qnt Q1
501.35 → 126.0 501.35 → 466.2 517.32 → 500.2
76 76 76
10 10 10
49 23 13
8 14 16
M + 2*AA
Q2 Qnt Q1
517.32 → 482.2 517.32 → 464.2 550.26 → 221.0
76 76 46
10 10 10
17 25 27
14 14 18
M + AA
Q2 Qnt Q1
550.26 → 73.0 550.26 → 533.3 533.25 → 516.2
46 46 76
10 10 10
89 9 15
12 16 16
Q2 Qnt Q1
533.25 → 498.2 533.25 → 480.3 352.27 → 317.1
76 76 41
10 10 10
19 25 19
14 14 8
Q2 Qnt
352.27 → 299.1 352.27 → 335.2
41 41
10 10
21 13
8 10
C24H40O4
392.57
Q1
410.18 → 81.0
21
10
65
12
Q2 Qnt Q1 Q2 Qnt Q1
410.18 → 91.1 410.18 → 357.2 426.31 → 355.3 426.31 → 391.3 426.31 → 373.3 426.29 → 373.2
21 21 41 41 41 56
10 10 10 10 10 10
99 19 25 13 17 19
14 10 10 12 10 10
Q2 Qnt Q1 Q2 Qnt
426.29 → 355.2 426.29 → 391.2 426.30 → 355.2 426.30 → 391.2 426.30 → 373.2
56 56 41 41 41
10 10 10 10 10
25 15 23 13 17
10 12 10 12 10
M + AA
M + AA
C24H40O5
408.57
M + AA
C24H40O5
408.57
M + AA
C24H40O5
408.57
M + AA
us
Taurodeoxycholic acid (TDCA)
515.70
8 10 8 18 18 16 6 8 12 16
an
C26H45NO7S
23 11 19 37 37 33 35 77 11 13
ed
Taurocholic acid (TCA)
499.70
CXP (V)
10 10 10
pt
C26H45NO6S
46 46 46 41 41 41 126
EP (V) CE (V) 10 10 10 10 10 10 10
126 126 71
ce
Taurochenodeoxycholic acid (TCDCA)
DP (V)
ip t
Progesterone
Fragments (m/z) Q1 336.34 → 283.2 Q2 336.34 → 319.2 Qnt 336.34 → 301.2 Q1 316.28 → 109.1 Q2 316.28 → 315.2 Qnt 316.28 → 97.0 Q1 430.41 → 83.1
cr
318.49
M+X+ (m/z) M + AA
M (g/mol)
M
Pregnanolone
Molecular formula C21H34O2
Compound
Q1 = qualifier 1, Q2 = qualifier 2, Qnt = quantifier, M + AA = ammonia - adduct, M + H+ = proton - adduct, DP = declustering potential, EP = entrance potential, CE = collision energy, CXP = collision cell exit potential
Page 19 of 21
22-S-OH-Chol 24-S-/25-OH-Chol 27-OH-Chol 7-OH-Chol CDCA/DCA Chol CA Desmosterol DOC GCDCA GCA GDCA GLCA GUDCA HDCA Pregnanedione Pregnanolone Progesterone Stigmasterol TCDCA TCA TDCA THDCA TLCA TUDCA T-α/β-MCA THDOC UDCA α-MCA β-MCA ω-MCA
0.999 0.997 0.996 0.999 0.999 0.991 0.999 0.958 0.999 0.998 0.999 0.999 0.999 0.998 0.994 0.995 0.998 0.999 0.997 0.999 0.999 0.999 0.999 0.998 0.998 0.999 0.996 0.996 0.999 0.999 0.999
y = 3,417x - 645 y = 1,888x - 80 y = 6,193x - 1,180 y = 964,882x - 27,753 y = 49,161x - 5,374 y = 3,831x + 2,253 y = 1E+06x - 56,934 y = 1,971x + 4,652 y = 1E+07x + 1E+06 y = 602,142x - 62,201 y = 1E+06x - 25,293 y =685,171x + 45,121 y = 515,926x - 3,676 y = 466,848x - 16,405 y = 12,264x + 6,158 y = 2.97E+06x + 79,433 y = 923,334x - 11,104 y = 3E06x + 15,618 y = 587x + 4,535 y = 1E+06x - 42,278 y = 1E+06x - 29,816 y = 803,720x - 21,728 y = 386,660x - 5,493 y = 3E+06x - 10,327 y = 694,933x - 20,462 y = 802,232x - 4,972 y = 2E+06x + 24,953 y = 124,576x + 3,094 y = 1E+06x - 19,493 y = 2E+06x - 970 y = 646,768x + 12,653
0.300 0.300 0.200 0.100 0.360 0.600 0.180 1.000 0.180 0.180 0.060 0.180 0.018 0.060 0.300 0.180 0.030 0.018 2.000 0.060 0.060 0.060 0.030 0.018 0.060 0.036 0.060 0.180 0.030 0.009 0.060
ed
pt
1,455 1,149 1,687 139,694 11,172 3,049 116,475 2,502 1,692,780 66,166 68,529 86,557 61,955 54,745 7,244 374,076 134,904 39,710 708 155,666 112,036 54,846 31,955 307,453 80,598 122,764 224,181 20,241 192,283 89,104 61,145
SD (µM)
CV (%) 0.43 0.61 0.27 0.14 0.23 0.80 0.11 1.27 0.11 0.11 0.06 0.13 0.12 0.12 0.59 0.13 0.15 0.01 1.21 0.12 0.11 0.07 0.08 0.12 0.12 0.15 0.12 0.16 0.14 0.05 0.09
21.7 20.5 28.7 13.0 9.7 20.3 9.0 26.8 9.6 9.3 6.3 10.7 16.0 11.8 11.9 10.7 17.1 10.8 30.8 12.6 10.9 6.9 9.7 16.1 11.7 10.2 11.9 13.8 16.1 13.1 9.4
Ac
ce
RSD (counts)
cr
LLoQ (µM)
us
Regression equation
an
R
M
Analytes
ip t
Table 2 Calibration curves and figures of merit of the baseline calibration
LLoQ = lower limit of quantification (µM) , RSD = residual standard deviation (counts), SD = standard deviation for the procedure (µM), CV = coefficient of variation (%)
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Table 3 Accuracy and precision for three concentration levels
Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ACC (%) Prec (%)
ip t
GCDCA C (µM) (%) 0,20 20,00 0,60 4,70 3,00 2,70 0,20 18,20 0,60 12,80 3,00 2,50
CA C (µM) (%) 0,20 19,20 0,60 17,10 3,00 3,30 0,20 3,70 0,60 3,90 3,00 0,40
Desmosterol C (µM) (%) 3,00 15,20 6,00 20,80 10,00 13,20 3,00 12,30 6,00 7,10 10,00 9,90
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
GCA C (µM) (%) 0,10 19,70 0,60 6,90 3,00 3,50 0,10 13,70 0,60 6,00 3,00 3,50
GDCA C (µM) (%) 0,20 19,40 0,60 10,50 3,00 6,60 0,20 16,80 0,60 6,10 3,00 5,90
GLCA C (µM) (%) 0,02 4,70 0,30 9,70 3,00 10,40 0,02 16,30 0,30 9,70 3,00 10,50
GUDCA C (µM) (%) 0,10 21,40 0,60 6,40 3,00 7,00 0,10 18,10 0,60 3,60 3,00 6,20
HDCA C (µM) (%) 0,90 12,50 9,00 6,80 15,00 4,20 0,90 20,00 3,00 6,10 15,00 4,20
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
Pregnanedione C (µM) (%) 0,20 18,40 0,60 11,60 3,00 3,50 0,20 11,90 0,60 13,00 3,00 2,80
Pregnanolone C (µM) (%) 0,03 17,20 0,60 8,60 3,00 8,00 0,03 18,90 0,60 11,50 3,00 8,50
Progesterone C (µM) (%) 0,20 11,90 0,60 7,80 3,00 5,80 0,20 11,50 0,60 13,40 3,00 5,60
Stigmasterol C (µM) (%) 2,00 16,10 6,00 6,70 10,00 7,20 2,00 55,60 6,00 15,90 10,00 15,50
TCDCA C (µM) (%) 0,10 16,10 0,60 6,70 3,00 7,20 0,10 16,70 0,60 8,00 3,00 8,10
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
TCA C (µM) (%) 0,10 19,00 0,60 7,30 3,00 5,90 0,10 17,70 0,60 6,30 3,00 5,80
TDCA C (µM) (%) 0,10 19,90 0,60 8,70 3,00 3,50 0,10 15,20 0,60 7,20 3,00 3,60
THDCA C (µM) (%) 0,03 16,10 0,60 6,40 3,00 3,70 0,03 19,00 0,60 5,30 3,00 3,70
TLCA C (µM) (%) 0,02 18,40 0,30 12,50 3,00 9,10 0,02 17,70 0,30 7,10 3,00 9,30
TUDCA C (µM) (%) 0,10 18,30 0,60 12,30 3,00 2,90 0,10 11,00 0,60 17,30 3,00 2,60
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
T-α/β-MCA C (µM) (%) 0,04 9,30 0,60 5,50 6,00 5,10 0,04 7,50 0,60 2,10 6,00 0,80
THDOC C (µM) (%) 0,10 22,00 0,60 19,00 3,00 3,60 0,10 17,10 0,60 17,30 3,00 3,10
UDCA C (µM) (%) 0,20 9,40 1,80 7,90 0,30 7,90 0,20 17,00 1,80 3,70 0,30 4,80
α-MCA C (µM) (%) 0,03 11,10 0,60 8,90 3,00 8,30 0,03 14,20 0,60 6,40 3,00 8,30
β-MCA C (µM) (%) 0,01 17,50 0,20 9,50 1,50 7,00 0,01 13,80 0,20 4,90 1,50 7,20
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
ω-MCA C (µM) (%) 0,10 18,70 C = concentration, ACC = accuracy, Prec = precision 0,30 6,30 3,00 5,10 0,10 17,90 0,30 5,30 3,00 5,20
an
us
cr
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
(%) 6,00 16,20 12,40 17,00 10,40 11,40
M
ACC (%)
DOC C (µM) (%) 0,20 11,00 0,60 4,80 3,00 5,00 0,20 14,30 0,60 4,40 3,00 5,00
Chol C (µM) 0,60 2,00 10,00 0,60 2,00 10,00
ed
Prec (%)
CDCA/DCA C (µM) (%) 0,20 19,80 0,60 3,70 3,00 4,40 0,20 14,30 0,60 1,10 3,00 5,10
pt
ACC (%)
7-OH-Chol C (µM) (%) 0,10 16,90 1,00 8,50 5,00 5,10 0,10 15,60 1,00 13,30 5,00 5,00
ce
Prec (%)
C 1 (LLOQ) C2 C3 C 1 (LLOQ) C2 C3
27-OH-Chol C (µM) (%) 0,20 19,00 1,00 19,20 2,00 23,30 0,20 39,80 1,00 23,00 2,00 28,80
Ac
ACC (%)
22-S-OH-Chol 24-S-/25-OH-Chol C (µM) (%) C (µM) (%) 0,30 10,10 0,60 18,00 3,00 12,00 3,80 12,20 5,00 13,50 6,30 11,00 0,30 29,10 0,60 25,20 3,00 16,20 3,80 11,90 5,00 14,10 6,30 11,10
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