Improving the Bioanalysis of Endogenous Bile Acids as Biomarkers for Hepatobiliary Toxicity using Q Exactive Benchtop Orbitrap?

Transcription

1 Troy Voelker, Mi Meg Tadem Labs, Salt Lake City, UT Kevi Cook, Patrick Beett Thermo Fisher Scietific, Sa Jose, CA Improvig the Bioaalysis of Edogeous Bile Acids as Biomarkers for Hepatobiliary Toxicity usig Q Exactive Bechtop Orbitrap? Itroductio Bile acids are produced i the liver by cytochrome P450-mediated oxidatio of cholesterol ad serve the purpose of emulsifyig dietary fats. Bile acids are also itegral to the elimiatio of cholesterol ad are ivolved i the regulatio of several biological processes. Bile acid levels i huma plasma ca be used as biomarkers for hepatobiliary toxicity ad thus aid i the idetificatio of harmful side effects of future therapeutics. Five bile acids [Cholic acid (CA), Cheodeoxycholic acid (CDCA), Taurocholic acid (TCA), Deoxycholic acid (DCA) ad Ursodeoxycholic acid (UDCA), [Figure 1] were idetified to moitor the bile acid levels i huma plasma. These bile acids were quatified usig LC-MS/MS ad also by high resolutio accurate mass HR/AM SIM mass spectrometry to obtai improved selectivity ad sesitivity. I order to circumvet the edogeous presece of bile acids i matrix, D4 stable labels of the CA, CDCA, TCA, DCA ad UDCA were used as surrogate aalytes to obtai statistical performace characteristics for the calibrator ad QC values to compare these techologies. FIGURE 1: Chemical Structures of CA, UDCA, CDCA, DCA ad TCA Troy Voelker et al. (2012) 1

2 Methodology 1. LC-MS/MS Mass Spec: Q Exactive or AB Sciex API 5000 Source ad ioizatio: ESI (egative io mode) Colum: C18, 30 x 3.00 mm Flow Rate: ml/mi Mobile Phase: A: Ammoium Acetate B: MeOH C: 10/90 water/mecn LC Program: Gradiet with colum backflush Source Temperature: 500 C MS Moitorig Parameters: SRM API 500 HR/AM SIM* Q Exactive CA 467.2g CA-D g UDCA 451.1g UDCA-D g CDCA 451.2g CDCA-D g DCA 451.3g DCA-D g TCA 514.1g TCA-D g *5ppm mass tolerace for XIC resolutio of 140k at m/z PPE Aliquot: 100 μl Huma Plasma (K EDTA) Precipitatio solvet: MeCN Recostitutio: 200 µl of 10 mm Ammoium Acetate: MeOH (50:50 v/v) Troy Voelker et al. (2012) 2

3 Results ad Discussios SIM, HR/AM SIM vs SRM Selected Reactio Moitorig (SRM) is the most commo method of performig quatitatio aalysis by mass spectrometry. Quatitative SRM is typically accomplished usig a triple quadrupole mass spectrometer capable of producig MS/MS fragmetatio. MS/MS is typically required because may compouds have the same itact mass while oly selected compouds have the precise MS/MS fragmetatio patter which ca be further isolated for selective quatitatio. Whe oly the first dimesio of MS is used to perform quatitatio (SIM), iterfereces will ofte arise from the lack of specificity, especially i complex biological matrices like plasma or whole blood. The secod dimesio of MS fragmetatio provides a uique fragmet for the compoud of iterest i a majority of cases, however, ucojugated bile acids represets a challege due to the istability of fragmets formed from the o-cojugated paret compouds. The combiatio of the specific precursor io mass/charge ad the product io mass/charge improves sigal-to-oise compared to SIM ad is used to selectively moitor for the compoud to be quatified. I Selected Io Moitorig (SIM) the mass spectrometer is set to sca multiple, selected masses; the arrower the rage the more specific the SIM plot. Oly compouds with the selected masses are detected, specificity is improved by applyig higher resolutio mass spectrometric aalysis. Although desired compouds ad uwated iterfereces ca have the same omial masses, their exact masses ofte differ by a fractio of a mass uit. Therefore, by couplig SIM with high resolutio, accurate mass (HR/AM) detectio, selectivity may be achieved for very complex matrices. METHOD DEVELOPMENT: Compoud tuig usig LC-MS/MS proved to be very difficult as 4 of the 5 bile acids did ot geerate product ios with appreciable yield for the desired aalytical sesitivity. Both APCI ad ESI were attempted over a rage of ph solvets. Precursor io itesities were observed facilitatig adequate sesitivity for measuremet of the desired assay LLOQ, however, additioal chromatographic resolutio was required to esure selective aalysis. I order to obtai improved selectivity, the acetate adduct of the precursor was selected ad the fragmeted to the ative precursor io for the SRM experimets. Separatio of the diastereomers UDCA ad CDCA proved to be easier tha the positioal isomers CDCA ad DCA. After fial chromatographic developmet, all five biles acids were baselie resolved usig a C18 3x30 mm colum ad eutral ph mobile phase withi a 6.5 miute cycle-time (Figure 8). Extractio recovery was greater tha 90% for all five bile acids. The method was validated uder curret regulatory guidelies. Troy Voelker et al. (2012) 3

4 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) DISADVANTAGES OF THE TRIPLE QUADRAPOLE MS (SRM Method): After our iitial method developmet ad aalysis with icurred samples, a differece i the backgroud oise respose for the ulabeled ative SRM trasitios relative to the stablelabeled aalog SRM trasitios was observed. High backgroud oise made itegratio of the ulabeled compouds difficult. Because the chromatographic backgroud usig the SRM method was extremely differet for the ulabelled ad labeled SRM trasitios, further LC method developmet was required i order to achieve a equivalet backgroud oise betwee the labeled ad ulabeled compouds (Figures 3 ad 4). Achievig ad demostratig mass balace was tedious. However, this process was required to esure that the respose of the surrogate stable-labeled compouds was equivalet to the respose of the ative ulabeled compouds i order to provide accurate cocetratio data for the study samples. Mass balace was achieved by ijectig two solutios cotaiig a test-mix of all the labeled ad ulabeled aalytes at both a high ad a low cocetratio. The solutios were ijected at =6 at each level ad the absolute respose of the labeled compouds was compared with the absolute respose of the ulabeled compouds. The accuracy of the solutios must be < 10% of each other ad the precisio () of the solutios must be less tha 15% before a ru ca be iitiated. TABLE 1: Itra assay Quality Cotrol Precisio (=6) o Q Exactive ad API 5000 High QC 400 g/ml (CA 200 g/ml) Bile Acid Q Exactive API 5000 CA UDCA CDCA DCA TCA Med QC 200 g/ml (CA 100 g/ml) Bile Acid Q Exactive API 5000 CA UDCA CDCA DCA TCA Low QC 3.00 g/ml (CA 1.50 g/ml) Bile Acid Q Exactive API 5000 CA UDCA CDCA DCA TCA Troy Voelker et al. (2012) 4

5 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) ADVANTAGES OF THE Q-EXACTIVE MS (HR/AM SIM Method) No Tuig Compoud tuig is ot required for the Q Exactive mass spectrometer ad data was acquired i targeted SIM mode. The eed to moitor the acetate adduct formatio was elimiated because the selectivity is based o the exact mass of the aalyte rather tha fragmet ios. Icreased Sesitivity The HR/AM SIM method provided improved sigal to oise with a stable ad oise-free baselie compared to the SRM method. HR/AM SIM improved sesitivity because there was o absolute sigal loss from fragmetatio of the precursor io. FIGURE 2: Chromatographic iterfereces i D 0 UDCA (Left) trasitios ot preset i D 4 UDCA (Right) trasitios with SRM for a extracted huma plasma sample Troy Voelker et al. (2012) 5

6 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) FIGURE 3: D 0 Chromatograms of UDCA, CA, DCA, CDCA, TCA ad aalog IS from extracted huma plasma ru o API FIGURE 4: Blak D 0 Chromatograms of UDCA, CA, DCA, CDCA, TCA ad aalog IS from extracted huma plasma ru o API Troy Voelker et al. (2012) 6

7 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) FIGURE 5: Blak, QCO ad LLOQ Chromatograms for D 4 -CA o API FIGURE 6: Blak, QCO ad LLOQ Chromatograms for D 4 -CA o Q Exactive. Troy Voelker et al. (2012) 7

8 Results ad Discussios (cotiued) FIGURE 7: Blak, QCO ad LLOQ Chromatograms for D 4 -UDCA o API FIGURE 8: Blak, QCO ad LLOQ Chromatograms for D 4 -CDCA o API Troy Voelker et al. (2012) 8

9 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) FIGURE 9: Blak, QCO ad LLOQ Chromatograms for D 4 -DCA o API FIGURE 10: QC0, Blak ad LLOQ Chromatograms for D 4 -UDCA/DCA/CDCA o Q Exactive Troy Voelker et al. (2012) 9

10 Results ad Discussios SIM, HR/AM SIM vs SRM (cotiued) FIGURE 11: Blak, QCO ad LLOQ Chromatograms for D 4 -TCA o API Figure 12: QC0, Blak ad LLOQ Chromatograms for D 4 -TCA o Q Exactive. Troy Voelker et al. (2012) 10

11 Coclusios A sesitive ad selective LC-MS/MS assay for the quatitatio of edogeous bile acids CA, CDCA, TCA, DCA, ad UDCA i huma plasma was re-developed o a Thermo Fisher Scietific Q Exactive HR/AM mass spectrometer. CA-d4, CDCA-d4, TCA-d4, DCA-d4, ad UDCA-d4 referece stadards were used as surrogate aalytes ad samples were extracted usig a protei precipitatio procedure. Liquid chromatography was optimized o a reversed phase colum with gradiet coditios ad 6.5 miute ru time. No tuig was required for the Q Exactive mass spectrometer ad data was acquired i selected io moitorig (SIM) mode. Troy Voelker et al. (2012) 11