Abstract

Abstract

Sensitivity, specificity and throughput are vital factors in bioanalytical laboratories. Most laboratories aim to achieve low levels of detection while using less sample and processing many samples per minute. These priorities can help increase efficiency and productivity in a therapeutic laboratory. Plate readers have commonly been used for drug screening but developing these methods can be time consuming. Furthermore, achieving high specificity with plate readers can be challenging, as can achieving high throughput with liquid chromatography methods. This technical note demonstrates a highly sensitive, highly specific, high-throughput quantitative solution for drug screening using the Zeno trap on the Echo^® MS+ system with ZenoTOF 7600 system.

Key-features

Key features of the Zeno MRM^HR scan with and without application of the Zeno trap

• Enhanced signal response: Achieve approximately 7x more signal for lidocaine and verapamil with the Zeno trap enabled compared to analysis without the Zeno trap
• Low detection limits: Detection of lidocaine and verapamil at 7.813 ng/mL was achieved with the Zeno trap
• Increased specificity:  The Zeno MRM^HR scan provided a full product ion scan from a targeted precursor analyte for a 1- second peak width and could be used for library searching to aid with compound confirmation^1,2
• High throughput: Rapid batch analysis of 63 samples in 2.6 minutes is possible
• Zeno MRM^HR method customization: Analyze a fixed fragment ion or scan a wide range of fragment ions

Introduction

Introduction

High-throughput screening labs commonly seek instruments with high sensitivity, high specificity and high throughput.³ Plate readers are often employed for early phase drug screening but development for these types of assays can be time consuming.⁴ Additionally, the endpoint of a plate reader assay is based on a reaction that generates an absorption or emission signal that is not caused by the compound itself, which can reduce the specificity of the assay.⁴

The Echo^® MS+ system with ZenoTOF 7600 system provides high-resolution mass spectral data, enhanced sensitivity with the application of the Zeno trap, high specificity and rapid throughput with sample ejection rates up to 1 sample/second.⁵ With the Zeno MRM^HR scan selected, the Echo^® MS+ system with ZenoTOF 7600 system can generate a single TOF MS spectrum and specific MS/MS product ion scans from targeted compounds with defined precursor masses. The user can opt to analyze a single product ion or opt to analyze a wide range of product ions at collision energies and collision energy spreads (a range of energies within a TOF MS/MS accumulation).

In this study, we demonstrated the ability of the Echo^® MS+ system with ZenoTOF 7600 system to produce full-range product ion scans in 1 second for 2 commonly used small molecule probes, lidocaine and verapamil. Nine different concentrations ranging from 7.813 ng/mL to 2000 ng/mL were analyzed using Zeno MRM^HR.

Methods

Methods

Sample preparation:  Concentration curves for lidocaine and verapamil were prepared in an Echo MS^® qualified 384-well plate using the serial dilution function of the INTEGRA (Hudson, NH) 8- channel pipettes. The samples contained lidocaine and verapamil at 2000 ng/mL, 1000 ng/mL, 500 ng/mL, 250.0 ng/mL, 125.0 ng/mL, 62.50 ng/mL, 31.30 ng/mL, 15.60 ng/mL and 7.813 ng/mL concentrations.

Acoustic ejection:  A total of 30 nL of the sample was ejected at an interval of 2 seconds per ejection using standard peak mode (400 Hz repetition rate). The carrier solvent consisted of a mix of 70:30 (v/v), water/acetonitrile and 0.1% (v/v) formic acid. A flow rate of 400 µL/min was used.

Mass spectrometry:  A Zeno MRM^HR method with a wide range of fragment ions was optimized and the “Zeno trapping” checkbox of the optimized method was then toggled “on” and “off” as the sample batches were run. The Zeno MRMHR method was optimized by directly infusing the 125 ng/mL standard of each compound via the Echo^® MS+ system (Tables 1-4).

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Data processing: Data were processed using the Analytics and Explorer modules of SCIEX OS software.

Increased-peak

Increased peak areas observed at all tested concentrations with the Zeno trap enabled

On average, with the Zeno trap enabled, 9.6-fold and 5.3-fold increases were observed for the signal-to-noise ratios for lidocaine and verapamil, respectively. These results were achieved when the Zeno MRM^HR scan was performed with a wide range of fragment ions (Figure 1).

Figure 1. Peaks observed with and without the Zeno trap enabled. Samples of 7.813 ng/mL lidocaine (A) and verapamil (B) were analyzed.

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The average area values for lidocaine and verapamil both increased at different magnitudes when the Zeno trap was enabled. These results are key at low concentrations, as the Zeno trap can therefore be used to enhance sensitivity (Figure 2).

Figure 2. Average peak area ratios for lidocaine and verapamil at low concentrations. The peak area ratio was determined by dividing the peak area observed with the Zeno trap enabled by the peak area observed without the Zeno trap. The ratio was calculated for lidocaine and verapamil at 3 low concentrations using a Zeno MRMHR method with a wide range of fragment ions.

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The calibration curves for both lidocaine and verapamil showed an increased slope with the Zeno trap enabled, indicating increased sensitivity for both analytes when using the Zeno trap (Figure 3). The peak area observed with the Zeno trap was significantly (p < 0.05) better than that without the Zeno trap (Table 5).

Figure 3. Calibrations curves for lidocaine (A) and verapamil (B). The calibration curves were constructed with and without use of the Zeno trap.

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Table 5. Calculated p values from the difference in peak areas with and without the Zeno trap enabled at 9 different concentrations.  Seven replicates were performed at each concentration.

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Accuracy and percent coefficient of variation (%CV) did not change significantly with and without the Zeno trap. However, no %CV or average accuracy values were reported for lidocaine at 7.813 ng/mL without the Zeno trap due to the lack of sensitivity. Analysis of 7.813 ng/mL lidocaine with the Zeno trap yielded a %CV of 6.7% and an average accuracy of 86.97% (Table 6).

Table 6. Average percent accuracy and %CV across all replicates of lidocaine and verapamil. Analysis was performed with and without the Zeno trap enabled at 9 different concentrations.

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Discussion

Discussion

When scanning a range of product ions, it is possible to quantify more than 1 product ion. By inputting 1 Zeno MRM^HR channel in the "MS Method" of SCIEX OS software, the user can select multiple fragment ions to quantify in the Analytics module (Figure 4). This experiment can be performed efficiently at rates as fast as 1 sample/second and is compatible with batch processing.

Figure 4. Processing results in SCIEX OS software for lidocaine and verapamil. A) Traces are shown for 250 ng/mL samples of lidocaine (blue) and verapamil (pink) based on a wide range of product ions from the MRMHR scan. Peaks span 1 second of data. B) Product ion scans are shown for lidocaine and verapamil. C) The quantitation table showing multiple product ions, derived from the Zeno MRMHR scan, for both lidocaine and verapamil.

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The area values significantly increased when the Zeno trap feature was applied to the Zeno MRM^HR scan. The Zeno trap routinely improved lower limits of quantitation by approximately 7-fold. The amount of increase appeared to be analyte-dependent, related to the m/z of the fragments.

Conclusion

Conclusion

• Use of the Zeno trap yielded approximate 6- and 9-fold increases in signal for verapamil and lidocaine, respectively
• Linearity was observed from 7.813 ng/mL to 2000 ng/mL when using the Zeno trap
• Analysis of 63 samples was completed in 2.6 minutes using the Zeno MRM^HR scan
• A full TOF MS scan was performed and a wide range of product ions were analyzed for verapamil and lidocaine using the Zeno MRM^HR scan

References

References

1. Extending Metabolome Coverage. SCIEX technical note, RUOMKT-02-7169-A.
2. Automated Targeted Screening of Hundreds of Metabolites. SCIEX technical note, RUO-MKT-02-2201-B.=
3. Rolf Kern and Chang Liu. True high throughput bioanalysis using the Echo^® MS System. SCIEX technical note, RUO-MKT02-11649-A.
4. Rapid MS/MS analysis with Acoustic Ejection Mass Spectrometry (AEMS). SCIEX technical note, RUO-MKT-02- 11385-A.
5. Highly sensitive quantification and selective identification of pesticides in food with Zeno MRMHR. SCIEX technical note, RUO-MKT-02-13902-C.