Important Aspects concerning the Quantification of Biomolecules by Time-of-Flight Secondary-Ion Mass Spectrometry

Abstract

Fundamental aspects regarding the use of time-of-flight secondary-ion mass spectrometry (TOF-SIMS) as a quantitative tool for the analysis of organic compounds are reported. The following factors are discussed: (1) the use of Poisson's law to correct for dead-time in single-ion data collection; (2) practical considerations concerning the analysis of “real world” samples; and (3) the effect of the etching process on the reproducibility of the intensity ratio (analyte/internal standard) of Ag-cationized species. To evaluate the importance of these factors, we used cocaine and cyclosporin A (CsA) as analytes because they show protonated and Ag-cationized species, respectively, in their SIMS spectra. Correction for detector dead-time using Poisson's law of single-ion counting expanded the dynamic range for cocaine by ∼2 orders of magnitude. For analyses requiring only a small dynamic range (i.e., CsA), the correction improved the % RSD of the slope from 2.43 to 0.87%. The maximum secondary-ion (SI) yield of CsA (Ag-cationized species) occurs at a CsA concentration ∼3 orders of magnitude higher than the therapeutic levels in blood (25–2000 ng/mL). It is discussed how this problem should be addressed. Analysis of variance (ANOVA) indicates that Ag substrates must be etched under identical conditions to obtain quantitative results when species requiring cationization are being analyzed.

Application of secondary ion and matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry for the quantitative analysis of biological molecules

“…In contrast, the CsA system (i.e., where the substrate is crucial for ionization) indicated that there was no sampling error for Ag substrates etched under identical conditions, but that there was a sampling error for Ag etched under similar conditions (107). Because the peak intensities of the Ag-cationized CsA species are substantially lower than those for cocaine, the precision of a peak area measurement is poorer.…”

“…This increase is followed by an exponential decrease in the CsA SI yield when the concentration is increased from 0.5 mg/mL to 80 mg/mL (region 11). to 0.5 mg/mL (region I), and steadily decreases throughout region 11 (107). Several factors can be considered to play a role in determining the shape of these molecular SI yield curves.…”

“…To better understand how the etching of Ag substrates affects quantitative TOF-SIMS measurements, a systematic study was carried out using two different biomaterials (84,107). The first system was a protonated molecular species, where the role of the substrate is to support and subsequentially desorb the analyte upon ion impact; cocaine hydrochloride was used as a model compound.…”

“…To determine how matrix effects from real biological samples influence surface coverage, the CsA system (vide infra) was used (107). The therapeutic range of CsA is 25-2000 ng/mL in whole blood, and is ca.…”

“…(107). These curves span four orders of magnitude (10 ng/mL -100 pg/mL), and it can easily be seen that without the use of the Poisson's law, linearity and point-to-point relative standard deviation (RSD) are poor; the latter results from the different absolute intensities, creating different degrees of detector deadtime.…”