( ...)Improved Quantitative Selectivity of Clenbuterol in Human Urine using ,,, High Resolution on the TSQ Quantum Mass Spectrometer

Mark Churchill¹, Mark Harrison¹, John Henninge,² and Hanne Lund²

1 Thermo Finnigan, Stafford House, 1 Boundary Park, Boundary Way, Hemel Hempstead, HP2 7GE, UK;
2 Aker University Hospital, Hormone Laboratory, Section for Doping Analysis,Trondheimsveien, N-0514, Oslo, Norway

Monitoring Clenbuterol in Humans

The data presented here was acquired on a Thermo Finnigan TSQ Quantum mass spectrometer Introduction Clenbuterol (Figure 1) is a beta-2-adrenergic agonist, an effective bronchodilator drug used for the treatment of human asthma. Clenbuterol also relieves bronchial airway smooth muscle contractions caused by Chronic Obstructive Pulmonary Disease (COPD) and allergyinduced respiratory distress.

Clenbuterol also has significant anabolic effects and could be used as a drug of abuse in athletes and livestock for its muscle growth stimulant properties. It also raises the body temperature and hence facilitates fat tissue catabolism. Due to Clenbuterol having these anabolic properties, it must be routinely monitored in biological samples by veterinary and human doping control laboratories. Figure 1: Clenbuterol Goal One of the limitations to quantitation is the unequivocal identification of analytes in biological samples due to endogenous matrix interferents.

This report describes the use of high resolution on the TSQ Quantum to exploit the negative mass defect of a compound containing Chlorine, such as Clenbuterol, and hence improve the selectivity of the quantitative assay. TSQ Quantum Tune View Optimisation of Clenbuterol Clenbuterol (C₁₂H₁₈C_l2N₂O, molecular weight 276.08) was infused, 0.1 ng/L, into the ESI source and the four most abundant product ions for the MS/MS breakdown were determined using the automated compound optimisation procedure in the TSQ Quantum (Figure 2).

The transition yielding the most abundant product ion (m/z 203.0) was selected for the analysis of Clenbuterol. Figure 2: Automated optimisation of MS/MS parameters for Clenbuterol Experimental Conditions Sample Preparation: Human urine extracts were prepared using a Solid Phase Extraction technique (Varian Bond-Elut LRC-18, methanol as eluent). The extracted urine was spiked with Clenbuterol in the concentration range 0.1, 0.5, 1, 5, 10, 50 and 100 pg/L for the calibration standards. No internal standard was used in this study.

Sample Analysis: The spiked urine extracts were chromatographed (Thermo Finnigan Surveyor System) on a C18 Elite 100 mm 2.1 mm column (Thermo Hypersil-Keystone) at a flow rate of 300 L/min with a linear gradient of 10% solvent B (Methanol/Ammonium acetate [10mM] 90/10 v/v) to 100% B over 5 minutes. Solvent A was Ammonium acetate (10 mM). The calibration standards were injected in duplicate at volumes of 10 L. MS Conditions: Mass spectrometer: Thermo Finnigan TSQ Quantum Ionisation mode: Electrospray (ESI), positive ion SRM: Clenbuterol 277.1 > 203.0 +/- 0.3 Da, 22 V Collision energy Resolution: Experiment 1 0.7 Da FWHM on Q1 and Q3
Experiment 2 0.1 Da FWHM on Q1, 0.7 Da FWHM on Q3 Two separate quantitative analyses were performed at peak widths of 0.1 Da and 0.7 Da Full Width Half Maximum (FWHM) on Q1 in SRM mode. A peak width of 0.7 Da FWHM was used on Q3 for all analyses. Results The chromatogram of a pure standard of Clenbuterol in aqueous solvent demonstrates the retention time at 5.8 minutes (Figure 3). Figure 3: Determination of Clenbuterol retention time Experiment 1: Quantitative Analysis Performed at 0.7 Da FWHM The data below shows the quantitative analysis of Clenbuterol in Human urine at peak width settings of 0.7 Da FWHM on Q1 and Q3. Chromatograms are shown for blank urine (Figure 4) and urine containing Clenbuterol at 0.1 pg/L (Figure 5). Figure 4: Urine blank, 0.7 Da FWHM

Figure 5: Clenbuterol, 0.1 pg/L in urine, 0.7 Da FWHM Experiment 1: Quantitative Analysis Performed at 0.7 Da FWHM A calibration curve of Clenbuterol analysed at 0.7 Da FWHM was constructed using linear fit of peak area plotted against concentration, weighted 1/X (Figure 6). A correlation coefficient of r2=0.9990 with an equation of Y = 8496.82+266143*X was obtained for the curve. Figure 6: Clenbuterol curve at 0.7 Da FWHM The peak area, back-calculated values and precision of all calibration standards are shown in Figure 7. Figure 7: Calculated standards at 0.7 Da FWHM Experiment 2: Quantitative Analysis Performed at 0.1 Da FWHM The data below shows the quantitative analysis of Clenbuterol in Human urine at peak width settings of 0.1 Da FWHM on Q1 and 0.7 Da FWHM on Q3. Chromatograms are shown for blank urine (Figure 8) and urine containing Clenbuterol at 0.1 pg/L (Figure 9). Figure 8: Urine blank, 0.1 Da FWHM

Figure 9: Clenbuterol, 0.1 pg/L in urine, 0.1 Da FWHM Experiment 2: Quantitative Analysis Performed at 0.1 Da FWHM A calibration curve of Clenbuterol analysed at 0.1 Da FWHM was constructed using linear fit of peak area plotted against concentration, weighted 1/X (Figure 10). A correlation coefficient of r2=0.9994 with an equation of Y = 2661.76+85951.1*X was obtained for the curve. Figure 10: Clenbuterol curve at 0.1 Da FWHM The peak area, back-calculated values and precision of all calibration standards are shown in Figure 11. Figure 11: Calculated standards at 0.1 Da FWHM Discussion Analysis, in SRM mode, of the spiked urine samples at a resolution setting of 0.7 Da FWHM resulted in a Clenbuterol peak eluting from the column upon a broad chemical noise background signal containing interferent peaks from the urine.

The same urine samples analysed at a peak resolution setting of 0.1 Da FWHM resulted in elimination of the interfering isobaric mass peaks and the broad background chemical noise previously seen in the analysis at a peak width setting of 0.7 Da FWHM. The selected reaction monitoring analysis performed at a higher resolution setting of 0.1 Da FWHM resulted in increased selectivity of the assay and hence an increase in the precision could be achieved.

The increase in selectivity at a peak width setting of 0.1 Da FWHM is due to the fact that Clenbuterol is a chlorinated compound and thus the negative mass deficiency can be used to eliminate interferents from the urine matrix in SRM mode. This increased selectivity can be achieved without detrimental loss of transmission. Typically only a factor of two to three fold decrease in peak area is observed between analyses performed at 0.7 and 0.1 Da FWHM, however, greater selectivity could then be achieved.

The calibration curves for Clenbuterol concentrations of between 0.1 to 100 pg/L at resolution settings of 0.1 and 0.7 Da FWHM both demonstrate excellent linearity. The calibration line at 0.7 Da FWHM showed a high intercept due to chemical background in the urine blank. This was significantly reduced by the use of high resolution.

The use of higher resolution to increase selectivity and precision could enable the limit of quantitation of an assay to be lowered and achieves a higher degree of confidence in identification of analytes in biological matrices.
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