The fast-evaporation method of sample preparation has been applied for quantitative analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. An instrumental protocol focusing on improvement of shot-to-shot repeatability and compensation for signal degradation has been developed for quantification of angiotensin II using the fast-evaporation technique and an internal standard. The fast-evaporation method was compared to the standard method of sample preparation (using a multicomponent matrix) in the quantitative analysis of angiotensin II, and found to be superior in several respects. Improvement in sample homogeneity using the fast-evaporation method enhanced both point-to-point repeatibility and sample-to-sample reproducibility. The relative standard deviations of the analyte/internal standard ratios (point RSD) were decreased by a factor of three compared to those obtained using the multicomponent matrix method. The average point RSD was found to be ca. 5% for the fast-evaporation technique. Two internal standards were evaluated for quantification of angiotensin II. The better one, 1-SAR-8-Ile angiotensin II, yielded a relative standard deviation of the standard curve slope of ca. 2.2% over two orders of magnitude of concentration (45 nM to 3000 nM), an improvement by a factor of two over the standard preparation method. Renal microdialysate samples, spiked with angiotensin II and the internal standard 1-SAR-8-Ile angiotensin II, were also analyzed using the fast-evaporation technique. The detection limit was calculated to be in the high attomole range (675 amol). Furthermore, the accuracy for a single determination of angiotensin II concentration in these samples was found to be 13.9% with a relative error of 8.19%.

Direct quantitative analysis using thin-layer chromatography (TLC) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI) has been demonstrated. An internal standard and a data collection protocol were used for analysis directly from TLC plates to compensate for shot-to-shot signal degradation, as well as deviations of analyte and internal standard spatial distributions within the TLC spot. Cocaine hydrochloride was used as a model compound for this study, and cocaine- d3 was used as the internal standard. Quantitative analysis by TLC/MALDI yielded comparable results to those obtained with stainless steel substrates (the standard MALDI method) for point-to-point repeatability, % RSD of the standard curve, and measurement precision. For silica gel and reverse-phase TLC plates, the relative standard deviation of the standard curve slope was better than 3%, the relative standard deviations of the analyte/internal standard intensity ratios ranged from 3.8% to 9.5%, the precision was estimated to be better than 12%, and the detection limits were estimated to be 60 pg for both TLC plate types. Quantitative analysis using stainless steel substrates yielded a lower detection limit than that obtained by TLC/MALDI by a factor of six. Perspectives for improving the detection limits of direct TLC/MALDI quantitative analysis are discussed. Index Headings: MALDI; TLC; Quantification.

Enhancement of ion intensity in static secondary-ionization mass spectrometry (SIMS) has been achieved by using a matrix-assisted sample preparation technique. Previous investigations of polymers and biomolecules by SIMS indicated that secondary-ion (SI) yield is dependent on substrate coverage. Recently we discovered a sample preparation technique that enhanced the SI yield of cyclosporin A (CsA) in an allograft patient sample and neat samples of CsA (1202 u) and polystyrene (M w=2650 u). The preparation technique involves deposition of a submonolayer of cocaine hydrochloride (5 µL of a 20-µg/mL MeOH solution) on an etched silver substrate, solvent evaporation, and subsequent deposition of the analyte. This preparation method resulted in ∼300% increase in the SI yield of CsA and polystyrene when deposited from neat solutions. The original discovery was observed when a blood extract that contained CsA was deposited on an etched Ag substrate that had been soaking in a dilute cocaine solution for ∼2 months. In these initial experiments, the SI yield of CsA was enhanced by over 1 order of magnitude.

Automation of data collection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry using a correlative analysis algorithm is demonstrated. This algorithm was employed to compensate for mass spectral jittering in MALDI data collection (e.g., peak shifts along the m/z axis, signal intensity deviations, etc.). Several important parameters for performing correlative analysis, such as the minimum correlation coefficient to be used and number of mass spectra to acquire prior to correlation, have been investigated and optimized. In addition, the correlation algorithm improved mass resolution of low- and high-molecular-weight compounds by as much as a factor of 4. Signal reproducibility in MALDI quantitative analysis also is improved when correlation is employed for data collection. This data collection algorithm can be used in conjunction with other instrumental optimization programs to allow for fully automated MALDI analysis, which is required for the routine applications carried out in many analytical laboratories.

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.