Some fundamental and technical aspects of the quantitative analysis of pharmaceutical drugs by matrix‐assisted laser desorption/ionization mass spectrometry

Abstract

The purpose of the present paper was to study some of the underlying physical and technical aspects of high-throughput quantitative matrix-assisted laser desorption/ionization (MALDI) of small drug molecules. A prototype MALDI-triple quadrupole instrument equipped with a high repetition rate laser was employed. Initially, the detection limits and dynamic ranges for the quantitation of four drugs (quinidine, danofloxacin, ramipril and nadolol) were determined. Internal standards were carefully chosen for each of these analytes in terms of structure similarity and fragmentation pathways. Three organic matrices were tested for these assays, resulting in different crystallization behaviors and measurement reproducibilities. a-Cyano-4-hydroxycinnamic acid yielded the best results and was subsequently employed for the quantitative determination of all four analytes. Further experiments considered the role of laser energy and pulse rate on the ablated areas as well as ion signals. Light microscope and scanning electron microscope images allowed the examination of the ablated area of the MALDI spots. The images showed convincing evidence that the ablated area was virtually void of crystals after analysis, with no preferential removal of material in the center of the laser's path. Average values for the amount of material ablated were determined to be 3.9 AE 0.5% of the total spot size, and as low as 19.5 attomoles of analyte were detectable for our most sensitive analyte, ramipril. It was calculated that, under these assay conditions, it was possible to accurately quantify less than 1 femtomole of all analytes with the use of appropriately pure internal standards. These studies showed very promising results for the quantitative nature of MALDI for small molecules with molecular weights less than 500 Da. Copyright # 2005 John Wiley & Sons, Ltd.Matrix-assisted laser desorption/ionization (MALDI) has recently seen an emergence in small molecule analysis, and appears extremely promising for high-throughput analysis and imaging studies. 1,2 One important factor that supported the implementation of MALDI in various applications was the development of atmospheric pressure (AP) MALDI. 3 AP-MALDI sources are relatively inexpensive and can be easily mounted onto several commercial liquid chromatography/mass spectrometry (LC/MS) instruments that are typically used for atmospheric pressure ionization (API) techniques, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). MALDI has several advantages over these techniques. Most importantly, MALDI can achieve a high sample throughput, 1,4 making it attractive to analytical laboratories for increasing productivity and efficiency. Furthermore, in comparison to ESI, MAL-DI is not as susceptible to ion suppression from salts or buffers. 5,6 As a result of these advantages, MALDI has recently been shown to permit extremely rapid and quantitative analyses for small molecules. 1,4,7 For the quantitative determination of low molecular weight compounds usin...

Design considerations for high speed quantitative mass spectrometry with MALDI ionization

“…Various MALDI matrices were tested, but ␣-cyano-4-hydroxycinnamic acid (CHCA) offered the best combination of wide applicability across the small molecule chemical space, homogenous crystallization and commercial availability [4,5]. The current experiments were performed with CHCA solutions (6.2 mg/ml, 36% methanol, 56% ACN, pH 2.5) available from Agilent Technologies (Palo Alto, CA) part number G2037A.…”

“…Assessments of the general utility of MALDI TOF for small molecule quantitative and qualitative analyses have been reported [2] and reviewed [3]. Studies of the practicality of the triple quadrupole approach for various applications have begun to appear in the literature using prototype triple quadrupole instruments [4,5], which have examined in detail the quantitative figures of merit of the technique in biological samples, emphasizing the importance of the use of internal standards. In a sister publication to this work [4], issues important to quantitation and general analytical utility such as chemical diversity, background produced by blank measurements, and s/n are covered in detail.…”

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

“…Sleno and Volmer [32] have also addressed some of the practical and theoretical aspects of quantification of low mass analytes by MALDI-TOF MS. They quantified quinidine, danofloxacin, ramipril and nadolol by MALDI and carefully selected the internal standards to match the structure and fragmentation pathways of the analytes.…”

4-Chloro-α-cyanocinnamic acid is an advanced, rationally designed MALDI matrix

“…The matrix exhibits a strong absorption at the laser wavelength used (typically 337 nm or 355 nm in the UV), which, after irradiation with a short pulse of laser light, leads to the ablation of a shallow surface region into the vacuum of the mass spectrometer, the release of intact gaseous matrix and analyte molecules, and their partial ionization. Since its introduction in the 1980s, MALDI has found widespread use in the mass spectrometric analysis of biological macromolecules but recently also has been used increasingly for small-molecule analysis (5,6).…”