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Zborník príspevkov z vedeckej konferencie - Department of ...

Zborník príspevkov z vedeckej konferencie - Department of ... Zborník príspevkov z vedeckej konferencie - Department of ...

analytika.sk
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structural information of the analytes using the power of fragmentation possibilities of the ion trap and also the gaining the high resolution and accurate masses measurement using time of flight analyzer. This work studied some potentialities of p(ITP-ITP)-HPLC/MS in the analysis of high molecular weight compounds (lysozyme served as model analyte) at low concentration level while present in the complex matrix (human saliva). In addition, selected mixture of discrete spacers was used in the p(ITP-ITP) stage of the combination, especially, to minimize very significantly complex character of the matrix and highly reproducible definition of the remaining compounds of the matrix as accompanied the analyte in the collected fractions. MS and MS/MS spectra, obtained from the reconstituted sample fractions, proved both the p(ITP-ITP) clean-up effect and ITP concentrating power for very low concentration levels of the analyte as present in complex biological matrix. EXPERIMENTAL Apparatus and HPLC-MS conditions Modified isotachophoretic analyzer ZKI-001 (Villa - Labeco, Spišská Nová Ves, Slovak Republic) in the columncoupling configuration of separation unit with the high voltage power supply capabling was used for preparative ITP experiments. The preseparation column of 1.8 mm I.D. (120 mm to detector) and the analytical column of 0.8 mm I.D. (160 mm to detector) were made of fluorinated ethylene-propylene copolymer (FEP). The applied driving currents were 600 A and 200 A in preseparation and analytical columns, respectively. Injection valve (44 l volume) of the sample loop and/or microsyringe (Hamilton) was used for the sample injection. On-column conductivity detectors were used for the detection of isotachophoretic zones. Preparative microfractionation valve with cca. 7 l volume of the inner loop was part of the analytical column. Concentrator 5301 (Eppendorf AG, Hamburg, Germany) was used for the lyofilization of the collected fractions. Electrolytes were filtered through 0.8 m membrane filter (Millipore, Molsheim, France) and stored in a fridge before analysis. HPLC-MS analyses of were performed by using Shimadzu LCMS-IT-TOF (Shimadzu, Kyoto, Japan). This MS analyzer is combining an electrospray ionization (ESI), a 3D quadrupol ion trap (IT) and an orthogonally accelerated time-offlight analyzer (TOF). HPLC experiments were performed on Reprosil-Gold 300 C18 column (100/2 mm; 5 m) (Dr.Maisch HPLC GmbH, Ammerbuch- Entringen, Germany) as using a gradient elution (water – acetonitrile) with a 0.2 ml/min flow rate: 0-1 min. - 10% ACN, 1-6 min. - 10-90%, 6.01-12 min. - 10% ACN. The column was thermostated to 40 °C. The MS conditions were as follows: electrospray capillary voltage +4.5 kV in positive ionization mode. Drying gas flow rate 10 L.min , drying gas temperature 200 °C. The MS–MS3 experiments automatically acquired data within 50-1500 m/z values in the positive mode. Fragmentation of ions in MS2 and MS3 experiments was performed using 25% energy and 25% of cooling gas with fragmentation ion window ±1,5 m/z. Data acquisition and evaluation was performed using LCMS Solution ver.3.4.151 (Shimadzu). Chemicals The electrolytes solutions were prepared from chemicals obtained from Merck (Darmstadt, Germany), Sigma-Aldrich (Steinheim, Germany) and Fluka (Buchs, Switzerland). All chemicals used were of analytical grade or additionally purified by the usual methods. Standard of discrete spacers (histidine, 2-Amino-2-hydroxymethyl-propane-1,3-diol, creatinine, - amino-n-caproic acid, - amino-n-butyric acid, – alanine) were purchased from Sigma. To minimize the problems with electroosmotic flow and convection of solution, both columns were filled with 1% (w/w) solution of hydroxyethylcellulose (high molecular weight anti convective agent) before pITP runs. Formic acid, acetonitrile (LC/MS quality) and water (LC/MS quality) were obtained from Merck (Darmstadt, Germany). For preparation of electrolytes and solutions of standards, water cleaned in 2 stages by Pro-PS unit (Labconco, Kansas City, U.S.A.) and Simplicity (Millipore, Molsheim, France) was used. Lysozyme standard (from chicken egg white) was obtained from Sigma and served as an internal standard. Standard solutions and sample preparation Saliva samples used as a matrix in the analyses of lysozyme were obtained from forth healthy volunteers (2 male and 2 female). Samples were filtered through 0.45 m syringe filter (Millipore) and 5 l of concentrated formic acid was added to 1 mL of sample immediately after obtaining. Lysozyme standard stock solution (1 mg/mL) was prepared by dissolving of 1 mg of standard in 1 mL of water (LC/MS quality). Working solution (c = 0.05 mg/L) was prepared by a proper dilution of lysozyme standard stock solution with water (LC/MS quality). The working solution was freshly prepared on each working day. The stock solutions of discrete spacers at 1×10 -2 mol/L concentration were prepared by dissolving of calculated amount in deionized water. The mixture of discrete spacers (HIS-histidine, TRIS-2-amino-2-hydroxymethyl-propane-1,3-diol, CREAT-creatinine, EACA- - amino-n-caproic acid, GABA- - amino-n-butyric acid, BALA- – alanine) was prepared from the stock solutions of spacers (10 -2 mol/L) by dilution with deionized water. The final concentration of each spacer in the mixture was 1 mmol/L. All fractions obtained by pITP were lyophilized (3 hours, 30C) after obtaining and reconstituted (89 L LC-MS water + 1 L 5% formic acid + 10 L 100% methanol) before the MS experiments. Zborník príspevkov z 18. medzinárodnej vedeckej konferencie "Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9 - 125 - hotel Falkensteiner, Bratislava 11. - 14. 10. 2010

RESULTS AND DISCUSSION HPLC-MS analysis of 5 l of lysozyme standard (cca 35 pmol was injected) is shown in Fig.1a. Elution position of lysozyme under our experimental conditions is at 8.2 minute showing a negative peak in TIC trace and it is marked by a bar. Averaged MS spectrum corrected for the background is shown in Fig.1b where several multiply charged ions over 800 m/z values are visible. After deconvolution of this spectrum quite nice peak of lysozyme is obtained showing molecular mass 14303.775 what is in very good agreement with the theoretical value 14303.8. HPLC-MS analysis of 10 ml of saliva sample is shown in Fig.2 where many high peaks are visible in TIC trace. Averaged MS spectrum corrected for the background is shown in Fig.2b from which it is clear that the main group of ions is shifted to lower m/z values (300-700). After deconvolution of this spectrum no peak of lysozyme is obtained what can be explained by the ion suppression as there are many ions present in the elution position of lysozyme. This fact is indicating that the direct HPLC-MS analysis is not possible to use for the analysis of lysozyme in saliva samples and some sample pretreatment technique has to be used before. 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 (x10,000,000) 1:TIC (1.00) 1.00 0.75 0.50 0.25 Inten.(x1,000) 14303.775 0.00 14050 14100 14150 14200 14250 14300 14350 14400 14450 14500 mass 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 1.5 1.0 0.5 Inten.(x10,000) 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z C) 1101.279 229.154 403.727 894.998 1192.943 299.102 340.173 520.851 654.277 744.905 847.415 1022.715 953.836 1301.401 545.736 1154.626 a) b) 1431.349 Fig. 1: TIC trace (a), MS spectrum (b) and deconvoluted MS spectrum (c) from HPLC/MS analysis of lysozyme standard. For details, see Experimental. Zborník príspevkov z 18. medzinárodnej vedeckej konferencie "Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9 - 126 - hotel Falkensteiner, Bratislava 11. - 14. 10. 2010

RESULTS AND DISCUSSION<br />

HPLC-MS analysis <strong>of</strong> 5 l <strong>of</strong> lysozyme standard (cca 35 pmol was injected) is shown in Fig.1a. Elution position <strong>of</strong><br />

lysozyme under our experimental conditions is at 8.2 minute showing a negative peak in TIC trace and it is marked by a bar.<br />

Averaged MS spectrum corrected for the background is shown in Fig.1b where several multiply charged ions over 800 m/z<br />

values are visible. After deconvolution <strong>of</strong> this spectrum quite nice peak <strong>of</strong> lysozyme is obtained showing molecular mass<br />

14303.775 what is in very good agreement with the theoretical value 14303.8. HPLC-MS analysis <strong>of</strong> 10 ml <strong>of</strong> saliva sample<br />

is shown in Fig.2 where many high peaks are visible in TIC trace. Averaged MS spectrum corrected for the background is<br />

shown in Fig.2b from which it is clear that the main group <strong>of</strong> ions is shifted to lower m/z values (300-700). After<br />

deconvolution <strong>of</strong> this spectrum no peak <strong>of</strong> lysozyme is obtained what can be explained by the ion suppression as there are<br />

many ions present in the elution position <strong>of</strong> lysozyme. This fact is indicating that the direct HPLC-MS analysis is not<br />

possible to use for the analysis <strong>of</strong> lysozyme in saliva samples and some sample pretreatment technique has to be used before.<br />

2.25<br />

2.00<br />

1.75<br />

1.50<br />

1.25<br />

1.00<br />

0.75<br />

0.50<br />

0.25<br />

(x10,000,000)<br />

1:TIC (1.00)<br />

1.00<br />

0.75<br />

0.50<br />

0.25<br />

Inten.(x1,000)<br />

14303.775<br />

0.00<br />

14050 14100 14150 14200 14250 14300 14350 14400 14450 14500 mass<br />

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0<br />

1.5<br />

1.0<br />

0.5<br />

Inten.(x10,000)<br />

0.0<br />

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z<br />

C)<br />

1101.279<br />

229.154<br />

403.727<br />

894.998<br />

1192.943<br />

299.102<br />

340.173<br />

520.851<br />

654.277<br />

744.905<br />

847.415<br />

1022.715<br />

953.836<br />

1301.401<br />

545.736<br />

1154.626<br />

a)<br />

b)<br />

1431.349<br />

Fig. 1: TIC trace (a), MS spectrum (b) and deconvoluted MS spectrum (c) from HPLC/MS analysis <strong>of</strong> lysozyme standard.<br />

For details, see Experimental.<br />

<strong>Zborník</strong> <strong>príspevkov</strong><br />

z 18. medzinárodnej <strong>vedeckej</strong> <strong>konferencie</strong><br />

"Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9<br />

- 126 -<br />

hotel Falkensteiner, Bratislava<br />

11. - 14. 10. 2010

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