Kinetic control of protonation in electrospray ionization.

The site of protonation in a molecule can greatly affect the fragments observed in product ion MS/MS spectra. In electrospray positive ionization mass spectra, protonation usually occurs predominantly on the most basic site on the molecule to produce the thermodynamically favored protonated species. However, the literature is unclear whether liquid phase or gas phase thermodynamics has the greater influence. This paper describes the protonation and fragmentation behavior of crizotinib and two of its impurities. Crizotinib has two possible protonation sites, a pyridine nitrogen and a secondary amine, piperidine nitrogen; the former is the favored site in the gas phase and the latter the more favored site in the liquid phase. The impurities contain alkyl substitution on the piperidine nitrogen, producing tertiary amine species. Literature precedence suggests that in the liquid phase, the piperidine nitrogen is still the most basic site but, in the gas phase, the pyridine nitrogen and the piperidine nitrogen have very similar basicities. Fragmentation data for the three molecules suggest that the secondary and tertiary amines protonate preferentially and almost exclusively on different sites. We propose that the secondary amine protonates on the piperidine nitrogen (influenced by solution thermodynamics) and the two tertiary amine structures protonate on the pyridine nitrogen because of steric hindrance at the most basic site of the molecule, allowing kinetic control of the protonation process.

Identification of multiple impurities in a pharmaceutical matrix using preparative gas chromatography and computer-assisted structure elucidation.

Gas chromatography (GC) with a preparative fraction collector (PFC) has been used to facilitate the identification of a number of volatile impurities at major and minor percentage levels in a pharmaceutical matrix by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The trapping process was optimized using liquid sorbents, and the impurities were trapped directly into a deuterated solvent. Challenges related to the pharmaceutical matrix were overcome by derivatization with boron trifluoride in methanol and extraction with heptane, producing the methyl esters of the carboxylic acid impurities and main component. GC coupled to atmospheric pressure chemical ionization mass spectrometry (APCI-MS) with a time-of-flight (TOF) detector was used to acquire accurate mass and isotopic data for the impurities, leading to the determination of their molecular formulas (MF). One dimensional (1D) and two-dimensional (2D) NMR experiments were also acquired to unambiguously determine the impurities' structure. The acquisition time of the latter experiments was minimized by using a high-resolution instrument equipped with a small (1.7 mm) cryogenic probe. The quality of the data was such that the structure of the impurities could be determined semiautomatically by using a computer-assisted structure elucidation (CASE) approach, even though the total amount of one of the isolated impurities was less than 60 nmol.

Direct analysis of pharmaceutical tablet formulations using Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging.

Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI MSI) has been used to directly analyse a range of tablets in order to assess the homogeneity of the active drug compound throughout the excipients contained within the tablets studied. The information gained from the imaging experiments can be used to improve and gain a greater understanding of the manufacturing process; such knowledge will enable improvements in finished product quality to make safer and more efficacious tablet formulations. Commercially available and prescription tablet formulations have been analysed, including aspirin, paracetamol, sildenafil citrate (Viagra(R)) and a batch of tablets in development (tablet X: placebo; 1 mg; 3 mg and 6 mg). MALDI MSI provides semi-quantitative information that is related to ion abundance, therefore Principal Component Analysis (PCA), a multivariate analysis technique, has been used to differentiate between tablets containing different amounts of active drug ingredient. Aspects of sample preparation have also been investigated with regard to tablet shape and texture. The results obtained indicate that MALDI MSI can be used effectively to analyse the spatial distribution of the active pharmaceutical component (API) in pharmaceutical tablet formulations.

Rapid, accurate and precise quantitative drug analysis: comparing liquid chromatography tandem mass spectrometry and chip-based nanoelectrospray ionisation mass spectrometry.

We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) system capable of achieving better than 2% accuracy, routinely over a wide concentration range of 1800 ng mL-1. We demonstrate that the necessary high precision, high accuracy and rapid analysis can be achieved using LC-MS/MS technology. Automated nanoelectrospray ionisation tandem mass spectrometry (nanoESI-MS/MS) technology can be employed to eliminate the chromatographic step completely. In this paper, nanoESI-MS/MS is evaluated and compared directly with LC-MS/MS for the quantitative analysis of two-test analytes, amitriptyline (ATT) and 5-methoxytryptamine (5-MTT), in aqueous/organic mixture. Calibration curves were found to be linear over a wide concentration range of 1800 ng mL-1 for both analytes using LC-MS/MS. Using nanoESI-MS/MS ATT gave a linear response while 5-MTT gave a non-linear response using nanoESI-MS/MS over the same concentration range as in LC-MS/MS. Accuracy and precision values of quality control samples (QCs) at four concentration levels were analysed in replicates of six at each level using 5-MTT and ATT as test analytes for both techniques. The LC-MS/MS system was capable of achieving accuracy levels of 99.50101.96% for ATT and 100.17100.40% for 5-MTT. Accuracy levels using nanoESI-MS/MS were not comparable to LC-MS/MS, they ranged from 90.09100.18% for ATT and 95.95113.55% for 5-MTT. The precision values obtained for nanoESI-MS/MS were in good agreement with those obtained by LC-MS/MS.

Clinical and local biological effects of an intratumoral injection of mda-7 (IL24; INGN 241) in patients with advanced carcinoma: a phase I study.

The melanoma differentiation-associated gene-7 (mda-7; approved gene symbol IL24) is a tumor suppressor gene whose expression induces selective apoptosis in tumor cells. To characterize the safety and biologic activity of mda-7 gene transfer, we conducted a phase I trial using intratumoral injections of an adenovirus containing the mda-7 construct (Ad-mda7; INGN 241; 2 x 10(10) to 2 x 10(12) vp) in 28 patients with resectable solid tumors. One hundred percent of injected lesions demonstrated INGN 241 vector transduction, transgenic mRNA, elevated MDA-7 protein, and apoptosis induction, with the highest levels near the injection site. Apoptosis of cells in injected tumors was consistently observed even in heavily pretreated patients. INGN 241 vector DNA and mRNA were detected more than 1 cm from the injection site, whereas MDA-7 protein and bioactivity were more widely distributed. Toxicity attributable to the injections was self-limiting and generally mild; however, one patient experienced a grade 3 SAE possibly related to the study drug. Evidence of clinical activity was found in 44% of lesions with the repeat injection schedule, including complete and partial responses in two melanoma patients. Thus intratumoral administration of INGN 241 is well tolerated, induces apoptosis in a large percentage of tumor cells, and demonstrates evidence of clinically significant activity.

Determination of pharmaceutical compounds in skin by imaging matrix-assisted laser desorption/ionisation mass spectrometry.

Matrix-assisted laser desorption/ionisation (MALDI) quadrupole time-of-flight mass spectrometry (Q-TOFMS) has been used to detect and image the distribution of a xenobiotic substance in skin. Porcine epidermal tissue was treated with 'Nizoral', a medicated shampoo containing ketoconazole (+/-)-1-acetyl-4-[p-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) as active ingredient. Following incubation for 1 h at 37 degrees C all excess formulation was washed from the surface. A cross-section of the drug-treated tissue was then blotted onto a cellulose membrane, precoated in matrix (alpha-cyano-4-hydroxycinnamic acid (CHCA)), by airspray deposition. In separate experiments the tissue surface was treated with Nizoral within a triangular former, and subsequently blotted onto a matrix-coated membrane. Sample membranes were then mounted into the recess of specialised MALDI targets with adhesive tape. All samples were analysed by MALDI-TOFMS using an Applied Biosystem 'Q-star Pulsar i' hybrid Q-TOF mass spectrometer fitted with an orthagonal MALDI ion source and imaging software. Detection of the protonated molecule was readily achievable by this technique. Treatment of the tissue within a template gave rise to images depicting the expected distribution of the drug, demonstrating that this technique is capable of producing spatially useful data. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross-sectional imprint of treated tissue. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Superimposing the mass spectrometric and histological images appeared to indicate drug permeation into the dermal tissue layer.

Quantitative determination of Piroxicam by TLC-MALDI TOF MS.

A quantitative thin-layer chromatography (TLC)-matrix-assisted laser desorption (MALDI) TOF mass spectrometry (MS) method for the determination of Piroxicam has been developed. Following preliminary experiments three different approaches to the incorporation of the internal standard (Tenoxicam) into the TLC plates were investigated. These were: (a) adding the internal standard to the mobile phase and pre-developing the plate, (b) coating the plate with internal standard by electrospraying prior to matrix application and finally, (c) mixing the internal standard into the matrix solution and electrospraying both. The most successful method was that where the internal standard was pre-developed over the plate. For this method linearity was observed over the range between 400 and 800ng of Piroxicam. The precision was found to be in the range of 1-9% R.S.D. from the average detected value (n = 5), dependent on the amount of analyte on the TLC plate. The proposed method was accurate with +/-2% deviation from the known amount of Piroxicam in the sample spot.

Thin-layer chromatography-matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry using particle suspension matrices.

Particle suspension matrices have been successfully utilized for the analysis of tetracycline antibiotics by thin-layer chromatography-matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (TLC-MALDI-TOF-MS). Particles of different materials and sizes have been investigated (Co-UFP, TiN, TiO2, Graphite and Silicon) by applying particle suspensions to eluted TLC plates. Mass spectra and mass chromatograms have been recorded directly from the TLC plates. Strong cationization by sodium and potassium was obtained in the positive ion mode, with [M+Na-NH3]+ ions being the predominant signals. The TLC-MALDI mass spectra recorded from graphite suspensions showed the lowest background noise and the highest peak intensities from the range of suspension matrices studied. The mass accuracy from graphite films was improved by adding the peptide Phe-Phe to the graphite suspensions. This allowed internal recalibration of the TLC-MALDI mass spectra acquired during a run. One major potential advantage of TLC-MALDI-TOF-MS has been demonstrated in the analysis of chlortetracycline and tetracycline in a mixture of oxytetracycline, chlortetracycline, tetracycline and minocycline. Examination of the TLC plate prior to MALDI analysis showed only an unresolved spot for chlortetracycline and tetracycline. However by investigation of the MALDI mass spectra and plotting of single ion chromatograms separate peaks for chlortetracycline and tetracycline could be obtained.

Thin-layer chromatography--postsource-decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small drug molecules.

The structural analysis of small drug molecules by directly coupling thin-layer chromatography (TLC) with postsource-decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is reported. The applicability of this technique is shown using two examples: the TLC-PSD MALDI analysis of two representatives of nonsteroidal antiinflammatory drugs (tenoxicam and piroxicam) and the analysis of the pharmaceutically active compound UK-137,457 and one of its related substances UK-124,912. The matrices alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and graphite are used to investigate the effect of the precursor ion selection on the TLC-PSD MALDI spectra of the drug molecules studied. Although alpha-CHCA enhances the [M+H]+ ion formation graphite produces in general only sodium adducts. Structural differentiation of tenoxicam and piroxicam is possible only by selecting the sodium adduct of both drug molecules as precursor ions. In the case of the TLC-PSD MALDI analysis of UK-137,457 and its related substance UK-124,912 at the 1% level, the PSD spectra obtained in alpha-CHCA by selecting the protonated adduct of the small molecules as precursor ions shows distinguishable dissociation patterns containing structurally significant information.