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Oil-Assisted Sample Preparation: A Simple Method for Analysis of Solid Samples Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Pui-Kin So and Zhong-Ping Yao* State Key Laboratory for Chirosciences and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administration Region, China
bS Supporting Information ABSTRACT: Common mass spectrometric techniques, e.g., electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), require samples to be soluble in suitable solvents. Samples with solubility problems have difficulties for their mass spectrometric characterization. In this paper, an oil-assisted sample preparation (OASP) method was introduced for the analysis of solid samples using MALDI-MS. The novel method involves the use of a droplet of oil (i.e., paraffin oil) as the mixing and loading media for solid analyte and solid matrix. Using this method, rapid on-target sample preparation can be easily achieved, and only a transferable minimal amount of analyte and matrix is required. This method was demonstrated to be applicable for a wide range of analytes, including poorly soluble organic compounds, polymers, organometallic compounds, membrane peptides, and biological solid samples. The novel method can also be used for the analysis of “wet” and solution samples. The limit of detection of the OASP MALDI-MS was determined to be 1 ng with reserpine.
’ INTRODUCTION Mass spectrometry (MS) is a rapid and sensitive tool for the characterization of various compounds. However, the analysis of samples with solubility problems is still a challenging task for mass spectrometry. These samples can be either insoluble or only soluble in solvents that are not compatible with MS analysis. Traditional ionization techniques such as electron ionization (EI) and chemical ionization (CI) can analyze samples in the solid state. However, these techniques require thermal vaporization of samples to the gas phase and, therefore, are not applicable for direct analysis of nonvolatile and thermally unstable compounds. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), which are two soft ionization techniques suitable for nonvolatile and thermally unstable samples, are conventionally based on solution samples and, therefore, require samples to be soluble in appropriate solvents. New techniques, including MALDI-MS-based techniques14 and ambient ionization techniques such as desorption electrospray ionization (DESI),5 laser-induced acoustic desorption coupled with electrospray ionization (LIAD/ESI),6 and direct analysis in real time (DART),7 have been explored for direct analysis of insoluble compounds by MS. The MALDI-MS-based techniques, which usually are called solvent-free MALDI-MS, have been more widely employed.810 The solvent-free MALDI-MS techniques were developed in early 200014 and the related sample preparation protocols have been further improved in recent years.1115 The commonly used protocols have been well-summarized in a recent publication by r 2011 American Chemical Society
Hughes et al.9 Briefly speaking, sample preparation for solventfree MALDI-MS analysis generally includes two steps: sample mixing and sample loading. The sample mixing step involves mechanically mixing of solid analyte and solid MALDI matrix, using a mortar and pestle or a mini-ball mill (MBM), or by vortex with the aid of metal balls.11,12 The sample loading step is to apply the solid analytematrix mixture to the MALDI target, usually by physically smearing a portion of the mixture onto the metallic target, using a microspatula.9,11,12 Using these sample preparation procedures, a wide range of solid samples, such as polymers,8,10 organometallic compounds,9 polycyclic aromatic hydrocarbons,1 organic pigments,16,17 and proteins and peptides,12,1820 have been successfully analyzed by MALDI-MS. Solvent-free MALDI-MS analysis has also been found to be advantageous over conventional solution-based MALDI-MS analysis in some respects, for example, solvent-driven degradation of samples can be prevented, matrix-sample segregation during crystallization can be avoided, and the choice of matrix is less restrictive.21,22 Although solvent-free MALDI-MS analysis is an effective technique for the analysis of insoluble samples, it has several drawbacks. First, compared to those for conventional MALDIMS analysis, the sample preparation procedures for solvent-free MALDI-MS analysis are more complicated and time-consuming. Received: January 30, 2011 Accepted: May 18, 2011 Published: May 18, 2011 5175
dx.doi.org/10.1021/ac2002593 | Anal. Chem. 2011, 83, 5175–5181
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Figure 1. MALDI-MS spectra of pigment Red 166 obtained by (a) the OASP method and (b) the solvent-free method.
Multisample preparation methods14 have been developed to allow the preparation of several samples at the same time for solvent-free MALDI-MS analysis, but special setup and extra consumables are required. Grinding solid analyte and solid matrix directly on the MALDI target has also been attempted;11 however, the quality of the mass spectra obtained was unsatisfactory. Second, it requires a relatively large amount of both analyte and matrix. Normally, at least ∼0.1 mg of analyte is required for effective mixing with the solid matrix, and, with a typical matrix:sample ratio ranging from ∼50:1 to 500:1 (w/w), a minimum amount of ∼5 mg of matrix is required for each analyte.1113 Third, sample loading of the solid analytematrix mixture onto the metallic target plate is somewhat difficult and has a high risk of contamination. The most common sample loading method involves physically crushing the mixed solid sample onto the target plate,9,11,12 which is a rather laborious task. Furthermore, since no adhesive medium is present between the solid sample and the target plate, there is a high risk of cross contamination of samples on the target plate and contamination to the ion source by the fallen-off solid samples.7,11 We consistently experienced that the sealing O-ring on the sample plate platform was contaminated with the fallen-off sample powders after some solvent-free experiments, resulting in breakdown of the instrument vacuum. Some other sample loading methods have also been used by some groups to solve this problem. These include: pressing the analytematrix mixture into a KBr disk and attaching it to the plate;2 suspending the mixture in a nonsolvent (e.g., water), loading the suspension on the plate, and allowing the nonsolvent to evaporate;3 and attaching the powder mixture to the plate with a double-sided adhesive tape.9 However, these methods have their own drawbacks, such as weak signal intensity
of molecular ions, difficulty in handling, poor spot-to-spot reproducibility, and the presence of interfering ion signals.3,9 Given the limitations described above, development of a simple, rapid, and economic sample preparation technique for MALDIMS analysis of solid samples is highly desirable. Here, we introduce an oil-assisted sample preparation (OASP) method for MALDI-MS analysis of solid samples. In this method, an oil-droplet is used to act as a medium for on-target mixing of analyte and matrix, bypassing the need for mixing processes in an another container, which is time-consuming and sample-consuming. Furthermore, the oil acts as an adhesive medium between the solid sample and the target plate, reducing the risk of cross contamination of samples and contamination of the flied-off sample powders to the ion source. In this paper, we demonstrate that this novel method allows MALDI-MS analysis of a wide range of poorly soluble samples and the sample preparation is simple, rapid, and required only a limited amount of samples.
’ EXPERIMENTAL SECTION Materials. R-Cyano-4-hydroxycinnamic acid (CHCA) and poly(methylmethacrylate) (PMMA) 2000 were purchased from Fluka. 2,5-Dihydroxy benzoic acid (DHB), paraffin oil, glycerol, petroleum jelly, reserpine, and sodium trifluoroacetate (NaTFA) were purchased from Sigma. 7,7,8,8-Tetracyanoquinodimethane (TCNQ) and vanadium(III) acetylacetonate (V(acac)3), as well as melamine and cyanuric acid, were purchased from Aldrich. Pigment Red 166 was purchased from Internal Laboratory USA (San Bruno, CA). Methanol was purchased from TEDIA (Fairfield, OH). 6,8-Dibromo-2-(40 -allyloxyphenyl)-4H-chromen-4-one (compound 1) was provided by Dr. K. F. Chan (ABCT, PolyU).23 5176
dx.doi.org/10.1021/ac2002593 |Anal. Chem. 2011, 83, 5175–5181
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Figure 2. MALDI-MS spectra of compound 1 obtained via (a) the OASP method and (b) the solvent-free method.
Sample Preparation for MALDI-MS Analysis. Oil-Assisted Sample Preparation (OASP) Method. First, a droplet of paraffin oil
or glycerol was spotted onto a sample well of the stainless steel target plate. With regard to the petroleum jelly, a minimal amount of the viscous solid was smeared onto a sample well to form a thin layer. Subsequently, a minimal and transferable amount (typically
