Quantitation of Alprazolam using Ambient Ionisation (DART) Mass Spectrometry
John H. Moncur1, Scott J. Campbell1 and Carsten Baessmann2
1 SpectralWorks Ltd, Runcorn, United Kingdom and 2 Bruker Daltonics GmbH & Co., Bremen, Germany
First presented as a poster at BMSS Ambient Ionisation SIG Meeting February 2024
Introduction
DART-MS lends itself to the fast screening and quantitation of drugs. However, this quantitation can present a number of challenges such as matrix effects, ionisation variability and data processing. A flexible software solution for sample splitting, calibration curve generation with multiple weightings, and report generation for unknowns is shown in this preliminary investigation. Height or area responses can be used as well as internal or external standard calibration and multiple m/z ratios if required. Accurate and reproducible quantitation of Alprazolam from 10 ppb to 10 ppm is shown using D5-Alprazolam internal standard using DART JumpShot® on a Bruker Impact II QTOF.
Methods and Materials
- DART JumpShot Pulsed Ionization System.
- Bruker Impact II QTOF MS.
- AnalyzerPro® XD Data Processing Software.
- Alprazolam solutions 10ppb, 50ppb, 100ppb, 500ppb, 1ppm, 5ppm, and 10ppm with 500ppb D-5 Alprazolam as internal standard.
- Alprazolam target m/z 309.0902.
- D5 Alprazolam target m/z 314.1215.
- Five replicates of each concentration level were pipetted on to a DART QuickStrip card and acquired in full scan mode.
- Final Sample was a blank (36 samples total).
- Internal and external standard calibration was performed using height and area responses. Equal, 1/x and 1/x2 weighting calculations were applied to the calibration curves.
Results
External Standard Calibration
Figure 2 shows a typical external standard calibration generated from the Alprazolam standards based on area response. Similar calibration responses were seen using response weightings of 1/x and 1/x2 from both the height and the area response data. This reflects the inconsistency in the absolute response from the DART ionisation.
Internal Standard Calibration
Figure 3 shows a typical internal standard calibration generated from the Alprazolam standards, using D-5 Alprazolam internal standard, based on area response. Similar calibration responses were seen using response weightings of 1/x and 1/x2 from both the height and the area response data. The internal standard calibration data was further investigated to show which calibration option gave the most accurate re-calculated amounts from the standard values.
Table 1 shows the R2 value and the mean absolute percent difference of the calculated amounts of the standard from the given standard values for the linear calibration curves with different weightings.
Table 2 shows the R2 value and the mean absolute percent difference of the calculated amounts of the standard from the given standard values for the quadratic calibration curves with different weightings.
The calibration curves for the Linear and Quadratic calculation, both with 1/x2 weighting, using area, are shown in Figures 4 and 5 respectively.
Conclusions
DART-MS quantitation for Alprazolam has been shown to be readily achievable. A number of calibration options were investigated. As well as the R2 value, the absolute average error across the concentration range was measured for each calibration option. The response over a range of 10ppb to 10ppm is not linear, however the use of weighted calibration was successful. For both Linear and Quadratic curve fitting, the 1/x2 weighting provided the smallest average error for each type. Of the two, the Quadratic fit with the 1/x2 weighting was the smallest error at an average of 9.0% and a R2 value of 0.998. The use of appropriate internal standards is essential.
The non-linearity of the calibration did not appear to be due to saturation of the MS detector and may have been due to an ionisation effect.
Further work to look at the effects of sample matrix is planned.