Studying Clenbuterol in Equine Plasma - Superior Ion Source Robustness & High Sensitivity

Robust performance and sustained high sensitivity are essential for the successful operation of a laboratory performing quantitative analysis. This article demonstrates the EVOQ LC-TQ’s sustained high-sensitivity performance in the analysis of equine plasma.

To assess the EVOQ LC-TQ’s ion source design, equine plasma was utilized in a dilute-and-shoot manner, thus eliminating the use of an in-line divert valve. The exposure of the ion source was done to all matrix eluting off the column during injection of every sample.

The de-solvation of the liquid eluent under the heated probe gas in the electrospray process causes the likely coalescence of contaminants such as proteins, sugars, lipids and salts with the ion source. This results in sensitivity degradation over time. The new ion source for EVOQ is designed by carefully examining the gas flow dynamics inside the ion source chamber, eliminating recirculation and avoiding areas of cold spots and coalescence.

Active Exhaust System

A gas entrainment exhaust (active exhaust) system is used to enhance the robustness of the ion source without compromising sensitivity. By slight pressure differential, the active exhaust system empties the ion source housing, thus avoiding recirculation of nebulized gases and contaminants. The nebulized components can be removed quickly thanks to the large exhaust opening, thus ensuring the cleanliness of the area facing the ion sampling orifice. The nebulized gases are entrained in the active exhaust system’s air-flow, enabling their removal from the LC-MS system.

Experimental

Sample Preparation

The matrix selected for the evaluation of the source robustness was crashed equine plasma spiked with clenbuterol. Cold acidified acetonitrile (3:1 v/v) was used for crashing equine plasma. The samples were centrifuged for 10 mins followed by the dispersion of the resulting supernatant with mobile phase A in 1:1 v/v.

Chromatography (Advance HPLC)

  • Column: C18, 3µ, 100x2.1mm (ACE, Part Number: 111-1002)
  • Flow rate: 0.45mL/min
  • Injection volume: 30µL
  • Mobile phase A: Water with 0.2% Formic acid
  • Mobile phase B: Acetonitrile with 0.2% Formic acid
  • Gradient conditions:
    • 0.0min 10% B
    • 0.3min 10% B
    • 2.5min 95% B
    • 2.8min 10% B
    • 4.5min 10% B

Mass Spectrometry (EVOQ Elite)

  • VIP Heated-ESI Temp: 300°C
  • Nebulizer gas: 90 units
  • Heated probe gas: 68 units
  • Cone gas: 10 units
  • Cone gas temp: 300°C
  • Spray voltage: +3500 V
  • Clenbuterol & d9-Clenbuterol MRM transitions:
    • Clenbuterol: m/z 277.1>168 (CE:25 v)
    • d9-Clenbuterol: m/z 286.1>204 (CE:12 v)

Results and Discussion

Four calibration curves were created and 100 matrix samples were injected between two adjacent calibrations. Maintaining a constant response factor (<10% RSD) for each calibration curve was the objective. The results yielded by EVOQ system surpassed the objective with the response factor RSD<5% with respect to a period of two days and more than 400 total injections of matrix samples.

The response factor is the ratio between the area ratio (calibrant/IS) and the concentration throughout the calibration range. The RSD% of response factor is a rigorous measure of sensitivity as it accounts every change in the calibration point on the curve. Any reduction in sensitivity will be shown by a higher %RSD response factor. It is essential to demonstrate robustness of ion source and sustained high sensitivity simultaneously. This demonstration has higher value when compared to a plot depicting repeated injections at high levels, typically spotted in vendors’ marketing literature.

The four calibration curves’ %RSD of the response factors was below 5%, a value well in line with the acceptance norms of validated bioanalytical techniques. Figure 3 depicts the overlaid first and fourth calibration curves, reflecting high sensitivity throughout the calibration range with insignificant divergence.

Overlay of curve 1 and curve 4, indicating sustained high sensitivity performance over the course of 400 plasma injections.

Figure 3. Overlay of curve 1 and curve 4, indicating sustained high sensitivity performance over the course of 400 plasma injections.

Tables 1 and 2 demonstrate the ion source’s sustained high sensitivity performance after repeatedly exposed to plasma samples, an important prerequisite for the adverse environment in a typical bioanalytical laboratory.

Table 1. . Calibration Curve no. 1

Sample ID Concentration (pg/mL) Area Ratio Response Factor
Plasma C1 5 0.0229 0.00459
Plasma C2 10 0.0468 0.00468
Plasma C3 25 0.1130 0.00452
Plasma C4 50 0.2162 0.00432
Plasma C5 100 0.4556 0.00456
Plasma C6 200 0.8706 0.00435
Plasma C7 400 1.7639 0.00441
Plasma C8 500 2.2001 0.00440
Plasma C9 750 3.5731 0.00476
Plasma C10 1000 5.0905 0.00509
Plasma C11 1500 6.9408 0.00463
Plasma C12 2000 9.5574 0.00478
Plasma C13 2500 11.6480 0.00466
Average: 0.004596
Std. Dev: 0.0002104
%RSD of Response FactorL 4.58%

Table 2. Calibration Curve no. 4

Sample ID Concentration (pg/mL) Area Ratio Response Factor
Plasma C1 5 0.0227 0.0045
Plasma C2 10 0.0477 0.0048
Plasma C3 25 0.1117 0.0045
Plasma C4 50 0.2144 0.0043
Plasma C5 100 0.4462 0.0045
Plasma C6 200 0.8549 0.0043
Plasma C7 400 1.7819 0.0045
Plasma C8 500 2.1869 0.0044
Plasma C9 750 3.5756 0.0048
Plasma C10 1000 4.9443 0.0049
Plasma C11 1500 6.7704 0.0045
Plasma C12 2000 9.5426 0.0048
Plasma C13 2500 11.8610 0.0047
Average: 0.004568
Std. Dev: 0.0002111
%RSD of Response Factor: 4.62%

The chromatograms for the first calibration level for each calibration curve are shown in figure 4.

Representative MRM chromatograms of the 5 ppt Clenbuterol in equine plasma (150 fg on-column) from the first to fourth calibrations, illustrating consistent peak areas, and thus response factors.

Figure 4. Representative MRM chromatograms of the 5 ppt Clenbuterol in equine plasma (150 fg on-column) from the first to fourth calibrations, illustrating consistent peak areas, and thus response factors.

Conclusion

In this experiment, the EVOQ LC-TQ successfully analyzed 400 matrix samples with insignificant sensitivity loss over a period of 48 hrs. The active exhaust design enables the ion source on the EVOQ LC-TQ to run at the highest sensitivity level in spite of repeated exposure to matrix, thus facilitating the successful operation of a quantitative analyses laboratory.

This information has been sourced, reviewed and adapted from materials provided by Bruker - Chemical And Applied Markets.

For more information on this source, please visit Bruker Daltonics.

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