Poor qualitative analysis of Naled necessarily leads to incorrect quantitative analysis.

This correspondence is in regards to a published article, titled "Prenatal Naled and Chlorpyrifos Exposure is Associated with Deficits in Infant Motor Function in a Cohort of Chinese Infants" Silver et al., Environ Int., 2017 Sep; 106:248-256. Upon careful review of this work we identified some significant issues in the mass spectral analysis of naled, specifically its identification and quantification. In this communication we address these issues and provide analytical data and rationale to support our criticism of the reported work. We collected mass spectral data for naled (analytical standard grade) under a variety of mass spectrometric conditions in an attempt to obtain a fragmentation pattern similar to what was reported in Silver et al., 2017. We however, could not reproduce a similar fragmentation pattern under any of the tested experimental conditions. Our results however were in excellent agreement with the National Institute of Standards and Technology (NIST) database, which is a very well established and widely accepted resource for such compounds. This leads us to conclude that naled was in all probability misidentified in the reported (Silver et al., 2017) study which consequently raises serious questions regarding the quantification of naled in the blood samples thus placing the whole statistical correlation of naled as a contributor to impairment of motor function in infants in question.

Laser desorption postionization mass spectrometry imaging of biological targets.

Laser desorption photoionization mass spectrometry (LDPI-MS) utilizes two separate light sources for desorption and photoionization of species from a solid surface. This technique has been applied to study a wide variety of molecular analytes in biological systems, but is not yet available in commercial instruments. For this reason, a generalized protocol is presented here for the use of LDPI-MS imaging to detect small molecules within intact biological samples. Examples are provided here for LDPI-MS imaging of an antibiotic within a tooth root canal and a metabolite within a coculture bacterial biofilm.

Ion sources for mass spectrometric identification and imaging of molecular species.

Covering: 2013 The ability to transfer molecular species to the gas phase and ionize them is central to the study of natural products and other molecular species by mass spectrometry (MS). MS-based strategies in natural products have focused on a few established ion sources, such as electron impact and electrospray ionization. However, a variety of other ion sources are either currently in use to evaluate natural products or show significant future promise. This review discusses these various ion sources in the context of other articles in this special issue, but is also applicable to other fields of analysis, including materials science. Ion sources are grouped based on the current understanding of their predominant ion formation mechanisms. This broad overview groups ion sources into the following categories: electron ionization and single photon ionization; chemical ionization-like and plasma-based; electrospray ionization; and, laser desorption-based. Laser desorption-based methods are emphasized with specific examples given for laser desorption postionization sources and their use in the analysis of intact microbial biofilms. Brief consideration is given to the choice of ion source for various sample types and analyses, including MS imaging.

Differentiation of microbial species and strains in coculture biofilms by multivariate analysis of laser desorption postionization mass spectra.

7.87 to 10.5 eV vacuum ultraviolet (VUV) photon energies were used in laser desorption postionization mass spectrometry (LDPI-MS) to analyze biofilms comprised of binary cultures of interacting microorganisms. The effect of photon energy was examined using both tunable synchrotron and laser sources of VUV radiation. Principal components analysis (PCA) was applied to the MS data to differentiate species in Escherichia coli-Saccharomyces cerevisiae coculture biofilms. PCA of LDPI-MS also differentiated individual E. coli strains in a biofilm comprised of two interacting gene deletion strains, even though these strains differed from the wild type K-12 strain by no more than four gene deletions each out of approximately 2000 genes. PCA treatment of 7.87 eV LDPI-MS data separated the E. coli strains into three distinct groups, two "pure" groups, and a mixed region. Furthermore, the "pure" regions of the E. coli cocultures showed greater variance by PCA at 7.87 eV photon energies compared to 10.5 eV radiation. This is consistent with the expectation that the 7.87 eV photoionization selects a subset of low ionization energy analytes while 10.5 eV is more inclusive, detecting a wider range of analytes. These two VUV photon energies therefore give different spreads via PCA and their respective use in LDPI-MS constitute an additional experimental parameter to differentiate strains and species.

Molecular imaging and depth profiling of biomaterials interfaces by femtosecond laser desorption postionization mass spectrometry.

Mass spectrometry (MS) imaging is increasingly being applied to probe the interfaces of biomaterials with invasive microbial biofilms, human tissue, or other biological materials. Laser desorption vacuum ultraviolet postionization with ∼75 fs, 800 nm laser pulses (fs-LDPI-MS) was used to collect MS images of a yeast-Escherichia coli co-culture biofilm. The method was also used to depth profile a three-dimensionally structured, multispecies biofilm. Finally, fs-LDPI-MS analyses of yeast biofilms grown under different conditions were compared with LDPI-MS using ultraviolet, nanosecond pulse length laser desorption as well as with fs laser desorption ionization without postionization. Preliminary implications for the use of fs-LDPI-MS for the analysis of biomaterials interfaces are discussed and contrasted with established methods in MS imaging.

Laser desorption VUV postionization MS imaging of a cocultured biofilm.

Laser desorption postionization mass spectrometry (LDPI-MS) imaging is demonstrated with a 10.5 eV photon energy source for analysis and imaging of small endogenous molecules within intact biofilms. Biofilm consortia comprised of a synthetic Escherichia coli K12 coculture engineered for syntrophic metabolite exchange are grown on membranes and then used to test LDPI-MS analysis and imaging. Both E. coli strains displayed many similar peaks in LDPI-MS up to m/z 650, although some observed differences in peak intensities were consistent with the appearance of byproducts preferentially expressed by one strain. The relatively low mass resolution and accuracy of this specific LDPI-MS instrument prevented definitive assignment of species to peaks, but strategies are discussed to overcome this shortcoming. The results are also discussed in terms of desorption and ionization issues related to the use of 10.5 eV single-photon ionization, with control experiments providing additional mechanistic information. Finally, 10.5 eV LDPI-MS was able to collect ion images from intact, electrically insulating biofilms at ~100 µm spatial resolution. Spatial resolution of ~20 µm was possible, although a relatively long acquisition time resulted from the 10 Hz repetition rate of the single-photon ionization source.

Feasibility of depth profiling of animal tissue by ultrashort pulse laser ablation.

Experiments were performed to examine the feasibility of mass spectrometry (MS) depth profiling of animal tissue by ~75 fs, 800 nm laser pulses to expose underlying layers of tissue for subsequent MS analysis. Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to analyze phospholipids and proteins from both intact bovine eye lens tissue and tissue ablated by ultrashort laser pulses. Laser desorption postionization mass spectrometry (LDPI-MS) with 10.5 eV single photon ionization was also used to analyze cholesterol and other small molecules in the tissue before and after laser ablation. Scanning electron microscopy was applied to examine the ablation patterns in the tissue and estimate the depth of the ablation craters. Ultrashort pulse laser ablation was found to be able to remove a layer of several tens of micrometers from the surface of eye lens tissue while leaving the underlying tissue relatively undamaged for subsequent MS analysis. MS analysis of cholesterol, phospholipids, peptides, and various unidentified species did not reveal any chemical damage caused by ultrashort pulse laser ablation for analytes smaller than ~6 kDa. However, a drop in intensity of larger protein ions was detected by MALDI-MS following laser ablation. An additional advantage was that ablated tissue displayed up to an order of magnitude higher signal intensities than intact tissue when subsequently analyzed by MS. These results support the use of ultrashort pulse laser ablation in combination with MS analysis to permit depth profiling of animal tissue.

Nanomole-scale protein solid-state NMR by breaking intrinsic 1HT1 boundaries.

We present an approach that accelerates protein solid-state NMR 5-20-fold using paramagnetic doping to condense data-collection time (to approximately 0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic (1)H T(1) longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional (13)C-(13)C and (13)C-(15)N solid-state NMR spectra for several to tens of nanomoles of beta-amyloid fibrils and ubiquitin in 1-2 d.