Analysis of low molecular weight compounds using MALDI‐ and LDI‐TOF‐MS: Direct detection of active pharmaceutical ingredients in different formulations

Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) is a high throughput, easy to use analytical technique. The simple sample preparation of this technique and its tolerance to the presence of contaminants are among its advantages. In contrast, depending on the matrix used, MALDI can ionize and generates ions in the low m /z range that complicate the interpretation of the spectra of low molecular weight compounds. To address this issue, one can envisage the use of tunable ionic matrices that can reduce the low m /z interferents. In this work, the ionic matrices triethylammonium α‐cyano‐4‐hydroxycinnamate and diisopropylammonium α‐cyano‐4‐hydroxycinnamate were used to directly analyze 14 pharmaceutical drugs in different formulations (coated tablets, noncoated tablets, capsules, and solutions). This methodology enabled the detection of their active compounds with minimum sample preparation, thus providing a straightforward approach for the forensic analysis of pharmaceutical drugs in the quest for detecting counterfeits. LDI‐MS experiments were also performed, and the active ingredient in all of the medicines analyzed were detected. However, MALDI‐MS spectra for the medicines analyzed herein showed less or no fragmentation than LDI‐MS, which makes the analysis easier.     

Improved spectra for MALDI MSI of peptides using ammonium phosphate monobasic in MALDI matrix

MALDI mass spectrometry imaging (MSI) enables analysis of peptides along with histology. However, there are several critical steps in MALDI MSI of peptides, 1 of which is spectral quality. Suppression of MALDI matrix clusters by the aid of ammonium salts in MALDI experiments is well known. It is asserted that addition of ammonium salts dissociates potential matrix adducts and thereafter decreases matrix cluster formation. Consequently, MALDI MS sensitivity and mass accuracy increase. Up to our knowledge, a limited number of MALDI MSI studies used ammonium salts as matrix additives to suppress matrix clusters and enhance peptide signals. In this work, we investigated the effect of ammonium phosphate monobasic (AmP) as alpha‐cyano‐4‐hydroxycinnamic acid (α‐CHCA) matrix additive in MALDI MSI of peptides. Prior to MALDI MSI, the effect of varying concentrations of AmP in α‐CHCA was assessed in bovine serum albumin tryptic digests and compared with the control (α‐CHCA without AmP). Based on our data, the addition of AmP as matrix additive decreased matrix cluster formation regardless of its concentration, and specifically, 8 mM AmP and 10 mM AmP increased bovine serum albumin peptide signal intensities. In MALDI MSI of peptides, both 8 and 10 mM AmP in α‐CHCA improved peptide signals especially in the mass range of m/z 2000 to 3000. In particular, 9 peptide signals were found to have differential intensities within the tissues deposited with AmP in α‐CHCA (AUC > 0.60). To the best of our knowledge, this is the first MALDI MSI of peptides work investigating different concentrations of AmP as α‐CHCA matrix additive to enhance peptide signals in formalin‐fixed paraffin‐embedded (FFPE) tissues. Further, AmP as part of α‐CHCA matrix could enhance protein identifications and support MALDI MSI‐based proteomic approaches.     

Identification and quantitative determination of atorvastatin calcium polymorph in tablets using FT-Raman spectroscopy

Atorvastatin calcium (ATC) is the active pharmaceutical ingredient (API) of the best selling lipid-lowering formulation Lipitor. Twelve ATC crystal forms are known and several pharmaceutical companies are developing or have developed generic drug formulations based on different ATC polymorphs. The strong overlap of the X-ray diffraction patterns (XRD) of the polymorphs with the respective patterns of the excipients, the presence of small API quantities in the tablet and the similarity of the crystal phase VIII XRD pattern used in the tablet examined in this work to that of phases IV and IX made identification difficult. Quantitative determination of ATC was attempted using Raman spectroscopy (RS), IR spectroscopy and X-ray powder diffraction. It was found that RS exhibited lower detection limit and a calibration model was constructed. Its application on commercial ATC tablets with 40mg strength yielded an error of 1.25%.     

Study of latent fingermarks by matrix‐assisted laser desorption/ionisation mass spectrometry imaging of endogenous lipids

Identification of suspects via fingermark analysis is one of the mainstays of forensic science. The success in matching fingermarks, using conventional fingermark scanning and database searching, strongly relies on the enhancement method adopted for fingermark recovery; this in turn depends on the components present in the fingermarks, which will change over time. This work aims to develop a robust methodology for improved analytical detection of the fingermark components. For the first time, matrix‐assisted laser desorption/ionisation mass spectrometry imaging (MALDI‐MSI) has been used to image endogenous lipids from fresh and aged, groomed and ungroomed fingermarks. The methodology was initially developed using oleic acid which was detected along with its degradation products over a 7‐day period, at three different temperatures in a time‐course experiment. The optimised methodology was then transferred to the imaging analysis of real fingermark samples. Fingermark patterns were reconstructed by retrieving the m/z values of oleic acid and its degradation products. This allowed the three aged fingermarks to be distinguished. In order to prove that MALDI‐MSI can be used in a non‐destructive way, a simple washing protocol was adopted which returned a fingermark that could be further investigated with classical forensic approaches. The work reported here proves the potential and the feasibility of MALDI‐MSI for the forensic analysis of fingermarks, thus making it competitive with other MSI techniques such as desorption electrospray ionisation (DESI)‐MS. The feasibility of using MALDI‐MSI in fingermark ageing studies is also demonstrated along with the potential to be integrated into routine fingermark forensic analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

A solvent‐free matrix application method for matrix‐assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI‐MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4‐bromophenyl‐1,4‐diazabicyclo(3.2.2)nonane‐4‐carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules. Copyright © 2010 John Wiley & Sons, Ltd.     

An On‐Tissue Paternò–Büchi Reaction for Localization of Carbon–Carbon Double Bonds in Phospholipids and Glycolipids by Matrix‐Assisted Laser‐Desorption–Ionization Mass‐Spectrometry Imaging

Matrix‐assisted laser desorption ionization mass spectrometry imaging (MALDI‐MSI) visualizes the distribution of phospho‐ and glycolipids in tissue sections. However, C=C double‐bond (db) positional isomers generally cannot be distinguished. Now an on‐tissue Paternò–Büchi (PB) derivatization procedure that exploits benzaldehyde as a MALDI‐MSI‐compatible reagent is introduced. Laser‐induced postionization (MALDI‐2) was used to boost the yields of protonated PB products. Collision‐induced dissociation of these species generated characteristic ion pairs, indicative of C=C position, for numerous singly and polyunsaturated phospholipids and glycosphingolipids in mouse brain tissue. Several db‐positional isomers of phosphatidylcholine and phosphatidylserine species were expressed with highly differential levels in the white and gray matter areas of cerebellum. Our PB‐MALDI‐MS/MS procedure could help to better understand the physiological role of these db‐positional isomers.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

Preparation and Characterization of Controlled-Release Floating Bilayer Tablets of Esomeprazole and Clarithromycin

Controlled-release effervescent floating bilayer tablets reduce dosage frequency and improve patient compliance with enhanced therapeutic outcomes. Generally, two different tablets of clarithromycin and esomeprazole, respectively, are given for the treatment of Helicobacter pylori infection and it might be worth incorporating both in a single tablet. In the current study, controlled-release floating bilayer tablets of clarithromycin and esomeprazole (F1–F4) were developed with different rates of polymeric materials by a direct compression method. During the formulation, Fourier-transform infrared spectroscopy (FTIR) analysis was performed for possible interactions between drugs and excipients. No interactions between drugs and excipients were noted. Moreover, the bilayer tablets’ thickness, diameter, friability, hardness, weight variation, dissolution, and percent purity were found within the acceptable limits. The floating lag time and total floating time of all formulations were found to be < 25 s and 24 h, respectively. The release of both the clarithromycin and esomeprazole started at the same time from the controlled-release floating bilayer tablets by anomalous non-Fickian diffusion, and the polymeric materials extended the drug release rate up to 24 h. In the case of F1, the results approached ideal zero-order kinetics. The dissolution profiles of the tested and reference tablet formulations were compared, but no significant differences were observed. It can be concluded that such controlled-release effervescent floating bilayer tablets can be efficiently used in clinical practice to reduce dosage frequency and increase patient compliance with continuous drug release for 24 h, which ultimately might enhance therapeutic efficacy.     

MALDIMSI在脂质组学研究中的应用

脂质组学概念自2003年被提出以来,其已成为研究生物体、组织或细胞中脂质的结构、功能及代谢途径的一门学科。脂质的种类众多,同时结构非常复杂,脂质的分析充满了困难和挑战。基质辅助激光解吸电离质谱成像(MALDI MSI)分析技术不仅可以进行物质鉴定,而且可对被分析物进行空间分布成像,近年来,该技术广泛地应用于脂质组学的研究。该文介绍了MALDI MSI在脂质组学研究中的样品处理、基质喷涂及应用方面的研究进展,并就目前存在的问题及解决方案进行了探讨,以期扩展MALDI MSI的应用范围。     

Effect of formulated ingredients on rapidly disintegrating oral tablets prepared by the crystalline transition method

The aim of this article was to determine the optimal ingredients for the rapidly disintegrating oral tablets prepared by the crystalline transition method (CT method). The effect of ingredients (diluent, active drug substance and amorphous sugar) on the characteristics of the tablets was investigated. The ingredients were compressed and the resultant tablets were stored under various conditions. The oral disintegration time of the tablet significantly depended on diluents, due to differences in the penetration of a small amount of water in the mouth and the viscous area formed inside the tablet. The oral disintegration time was 10-30 s for tablets with a tensile strength of approximately 1 MPa, when erythritol, mannitol or xylitol was used as the diluent. The increase in the tensile strength of tablets containing highly water-soluble active drug substances during storage was as large as that of tablets without active drug substances, while the increase in the tensile strength of tablets containing low water-soluble active drug substances was small. It was therefore found that highly water-soluble active drug substances were more suitable for the formulation prepared by the CT method than low water-soluble active drug substances. Irrespective of the type of amorphous sugar (amorphous sucrose, lactose or maltose) used, the porosity of tablets with 1 MPa of tensile strength was 30-40%, and their oral disintegration time was 10-20 s. The optimal ingredients for rapidly disintegrating oral tablets with reasonable tensile strength and disintegration time were therefore determined from these results.     

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3‐μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single‐pot solid‐phase‐enhanced sample preparation (SP3) method and analyzed by LC‐MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post‐translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system.     

In Vitro–In Vivo Correlation of Tianeptine Sodium Sustained-Release Dual-Layer Tablets

Tianeptine tablets are currently marketed to be designed for immediate-release tablets. The tianeptine has a short half-life, making it difficult to design for sustained-release tablets and achieve bioequivalence with the tianeptine immediate-release tablet (Stablon^®). We established the in vitro–in vivo correlation (IVIVC) of three formulations of tianeptine sustained-release tablets according to their granule size. To evaluate sustained drug release, in vitro tests were performed in pH 1.2 media for 24 h. In vivo pharmacokinetic analysis was performed following oral administration of reference drug and test drug to beagle dogs. The dissolution profile revealed delayed release as the size of the granules increased. The dissolution results were confirmed in pharmacokinetic analysis, showing that the half-life was delayed as granule size increased. The final formulation and reference drug showed an equivalent area under the curve (AUC). Through this, IVIVC was established according to the size of the tianeptine sodium granules, which is the purpose of this study, and was used to predict in vivo pharmacokinetics from the formulation composition. This approach may be useful for determining optimal formulation compositions to achieve the desired pharmacokinetics when developing new formulations.     

High‐Spatial Resolution Mass Spectrometry Imaging: Toward Single Cell Metabolomics in Plant Tissues

Mass spectrometry imaging (MSI) is a powerful tool that has advanced our understanding of complex biological processes by enabling unprecedented details of metabolic biology to be uncovered. Through the use of high‐spatial resolution MSI, metabolite localizations can be obtained with high precision. Here we describe our recent progress to enhance the spatial resolution of matrix‐assisted laser desorption/ionization (MALDI) MSI from ∼50 μm with the commercial configuration to ∼5 μm. Additionally, we describe our efforts to develop a ‘multiplex MSI’ data acquisition method to allow more chemical information to be obtained on a single tissue in a single instrument run, and the development of new matrices to improve the ionization efficiency for a variety of small molecule metabolites. In combination, these contributions, along with the efforts of others, will bring MSI experiments closer to achieving metabolomic scale.     

In vitro evaluation of mucoadhesive vaginal tablets of antifungal drugs prepared with thiolated polymer and development of a new dissolution technique for vaginal formulations

The main objective of this work was to develop antifungal matrix tablet for vaginal applications using mucoadhesive thiolated polymer. Econazole nitrate (EN) and miconazole nitrate (MN) were used as antifungal drugs to prepare the vaginal tablet formulations. Thiolated poly(acrylic acid)-cysteine (PAA-Cys) conjugate was synthesized by the covalent attachment of L-cysteine to PAA with the formation of amide bonds between the primary amino group of L-cysteine and the carboxylic acid group of the polymer. Vaginal mucoadhesive matrix tablets were prepared by direct compression technique. The investigation focused on the influence of modified polymer on water uptake behavior, mucoadhesive property and release rate of drug. Thiolated polymer increased the water uptake ratio and mucoadhesive property of the formulations. A new simple dissolution technique was developed to simulate the vaginal environment for the evaluation of release behavior of vaginal tablets. In this technique, daily production amount and rate of the vaginal fluid was used without any rotational movement. The drug release was found to be slower from PAA-Cys compared to that from PAA formulations. The similarity study results confirmed that the difference in particle size of EN and MN did not affect their release profile. The release process was described by plotting the fraction released drug versus time and n fitting data to the simple exponential model: M(t)/M(∞)=kt(n). The release kinetics were determined as Super Case II for all the formulations prepared with PAA or PAA-Cys. According to these results the mucoadhesive vaginal tablet formulations prepared with PAA-Cys represent good example for delivery systems which prolong the residence time of drugs at the vaginal mucosal surface.     

Application of transmission near-infrared spectroscopy to uniformity of content testing of intact steroid tablets

Transmission near-infrared (NIR) spectroscopy was used for the rapid and non-destructive determination of the content of a hormone steroid in single intact tablets. Tablets produced for clinical trial purposes containing 5, 10, 15, 20 and 30 mg (2.94, 5.88, 8.82, 11.76 and 17.64% m/m, respectively) were used to develop calibration models without the need to specially prepare any out of specification tablets. Reference values for the individual tablets used in the NIR calibration models and test set were measured by reversed-phase high performance liquid chromatography (HPLC). Partial least squares regression using standard normal variate transformed second-derivative spectra over the range 800 to 1040 nm gave the optimum calibration model with a standard error of calibration of 0.52 mg per tablet. Measurements of an independent test set gave comparable results (standard error of prediction 0.31 mg per tablet). Measurement errors for a single tablet (RSD < 2.5% for a given active level) were sufficiently small to allow the procedure to be applied to pharmacopoeial uniformity of content testing of batches of these tablets and permitted the non-destructive testing of 30 tablets in under 20 min as compared to 6 h by HPLC.     

In situ assessment of atorvastatin impurity using MALDI mass spectrometry imaging (MALDI-MSI)

The analysis of impurities and degradation products in pharmaceutical preparations are usually performed by chromatographic techniques such as high-performance liquid chromatography (HPLC). This approach demands extensive analysis time, mostly due to extraction and separation phases. These steps must be carried out in samples in order to adapt them to the requirements of the analytical method of choice. In the present contribution, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was employed to quantify an important degradation product in atorvastatin calcium 80 mg tablets: the atorvastatin lactone. Through the standard of the impurity, it was possible to perform quantitative analysis directly on the drug tablet, using a quick and novel approach, suitable for quality control processes in the pharmaceutical industry.     

Rapid analysis of controlled substances using desorption electrospray ionization mass spectrometry

The recently developed technique of desorption electrospray ionization (DESI) has been applied to the rapid analysis of controlled substances. Experiments have been performed using a commercial ThermoFinnigan LCQ Advantage MAX ion-trap mass spectrometer with limited modifications. Results from the ambient sampling of licit and illicit tablets demonstrate the ability of the DESI technique to detect the main active ingredient(s) or controlled substance(s), even in the presence of other higher-concentration components. Full-scan mass spectrometry data provide preliminary identification by molecular weight determination, while rapid analysis using the tandem mass spectrometry (MS/MS) mode provides fragmentation data which, when compared to the laboratory-generated ESI-MS/MS spectral library, provide structural information and final identification of the active ingredient(s). The consecutive analysis of tablets containing different active components indicates there is no cross-contamination or interference from tablet to tablet, demonstrating the reliability of the DESI technique for rapid sampling (one tablet/min or better). Active ingredients have been detected for tablets in which the active component represents less than 1% of the total tablet weight, demonstrating the sensitivity of the technique. The real-time sampling of cannabis plant material is also presented.     

A quantitation method for mass spectrometry imaging

A new quantitation method for mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) has been developed. In this method, drug concentrations were determined by tissue homogenization of five 10 μm tissue sections adjacent to those analyzed by MSI. Drug levels in tissue extracts were measured by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The integrated MSI response was correlated to the LC/MS/MS drug concentrations to determine the amount of drug detected per MSI ion count. The study reported here evaluates olanzapine in liver tissue. Tissue samples containing a range of concentrations were created from liver harvested from rats administered a single dose of olanzapine at 0, 1, 4, 8, 16, 30, or 100 mg/kg. The liver samples were then analyzed by MALDI-MSI and LC/MS/MS. The MALDI-MSI and LC/MS/MS correlation was determined for tissue concentrations of ~300 to 60,000 ng/g and yielded a linear relationship over two orders of magnitude (R(2) = 0.9792). From this correlation, a conversion factor of 6.3 ± 0.23 fg/ion count was used to quantitate MSI responses at the pixel level (100 μm). The details of the method, its importance in pharmaceutical analysis, and the considerations necessary when implementing it are presented.     

Some physical properties of tabletted seed of Garcinia kola (HECKEL)

The formulation of Garcinia kola seeds into tablet dosage form and evaluation of some physical properties of the tablets are presented. A chemical assay was conducted on the dry, powdered seeds as well as the crude aqueous extract of the seeds. The dry powdered seeds contain 0.003% of flavonoids while the crude extract contained 0.007% of flavonoids based on rutin used as the standard. The powdered material (50 mg) and crude extract (10 mg) were formulated into tablets using the wet granulation method. Named binders were evaluated in these formulations. The various tablet parameters were evaluated, namely: weight variation, thickness and diameter, hardness, friability, disintegration time, dissolution profile and content uniformity. The results indicated that the tablets had good disintegration time, dissolution and hardness/friability profiles. Tablets formulated with starch had the best disintegration properties but were consequently very friable. Tablets formulated from 10 mg of the crude extract needed a larger proportion of diluents, which affected the tablet properties.