Thermogravimetry combined with electrospray and atmospheric pressure chemical ionization mass spectrometry for characterization of synthetic polymers.

RATIONALE: Thermogravimetry (TG) combined with electrospray and atmospheric chemical ionization (ESI+APCI) mass spectrometry (MS) was developed to rapidly characterize thermal decomposition products of synthetic polymers and plastic products. The ESI-based TG-MS method is useful for characterizing thermally labile, nonvolatile, and polar compounds over an extensive mass range; and the APCI-based TG-MS counterpart is useful for characterizing volatile and nonpolar compounds. Both polar and nonpolar compounds can be simultaneously detected by ESI+APCI-based TG-MS. METHODS: Analytes with different volatility were produced from TG operated at different temperatures, which were delivered through a heated stainless-steel tube to the ESI+APCI source where they reacted with the primary charged species generated from electrospray and atmospheric pressure chemical ionization (ESI+APCI) of solvent and nitrogen. The analyte ions were then detected by an ion trap mass spectrometer. RESULTS: A semi-volatile PEG 600 standard was used as the sample and protonated and sodiated molecular ions together with adduct ions including [(PEG)n + 15]+ , [(PEG)n + 18]+ , and [(PEG)n + 29]+ were detected by TG-ESI+APCI-MS. The technique was further utilized to characterize thermal decomposition products of nonvolatile polypropylene glycol (PPG) and polystyrene (PS) standards, as well as a PS-made water cup and coffee cup lid. The characteristic fragments of PPG and PS with mass differences of 58 and 104 between respective ion peaks were detected at the maximum decomposition temperature (Tmax ). CONCLUSIONS: The information obtained from the TG-ESI+APCI-MS analysis is useful in rapidly distinguishing different types of polymers and their products. In addition, the signals of the additives in the polymer products, including antioxidants and plasticizers, were also detected before the TG temperature reached Tmax .

Measurement of poly(ethylene glycol) by cell-based anti-poly(ethylene glycol) ELISA.

Poly(ethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/alphaPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The alphaPEG cell-coated plate bound biotinylated PEG(5K) (CH(3)-PEG(5K)-biotin) and CH(3)-PEG(5K)-(131)I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5, 10, or 20 kDa) and a known amount of CH(3)-PEG(5K)-biotin allowed construction of a reproducible competition curve. The alphaPEG cell-based competition ELISA measured small molecules derivatized by PEG(2K), PEG(5K), PEG(10K), PEG(20K), and PEG(5K) at concentrations as low as 58.6, 14.6, 3.7, 3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the alphaPEG cell-based competition ELISA. Finally, we show here that the alphaPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG(5K), comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH(3)-PEG(5K)-(131)I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.

Study of human neutrophil peptides in saliva by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides - HNP 1, 2, and 3 - in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI-TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva-absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age-dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging.

Removal of Diethylhexyl Phthalate from Hands by Handwashing: Evidence from Experimental N-of-1 and Crossover Designs.

Phthalate exposure through skin is often neglected due to the small quantity and limited dermal absorption rate. However, free phthalate can be ingested by hand-to-mouth action or by contact with food. To evaluate the effectiveness in removing phthalate exposure on hand, we compare here the removal efficiency of di-(2-ethylhexyl)phthalate (DEHP) on hands by handwashing with soap-and-water versus water-only. In two three-day N-of-1 trials, residual DEHP was measured in a single female adult who washed exposed hands with soap-and-water or water-only. Subsequently, a crossover study was performed by randomly assigning another 28 subjects equally to wash with soap-and-water or with water-only, and then each one received the other treatment 24 hrs later. In the N-of-1 trials, mean DEHP removal rates range from 95.9% (SD = 0.1%) to 97.0% (SD = 2.5%) for soap-and-water handwashes, and 1.8% (SD = 0.1%) to 7.0% (SD = 0.3%) (n = 3) for water-only. In the crossover study, mean removal rate was 94.6% (SD = 6.5%) for handwashing with soap-and-water (n = 28) and 8.7% (SD = 5.7%) for water-only (n = 28). We concluded that handwashing with soap-and-water removes 80% more DEHP than handwashing with water alone, and may be a cost-effective way of removing other endocrine disruptors from hands.

Rapid identification of pesticides in human oral fluid for emergency management by thermal desorption electrospray ionization/mass spectrometry.

Self-poisoning with pesticides accounts for approximately one-third of all suicides worldwide. To expedite rescue in the emergency department, it is essential to develop a point-of-care analytical method for rapid identification of ingested pesticides. In this study, five of the most common pesticides ingested by self-poisoning patients in Taiwan were analyzed from oral fluid samples. Pesticide-oral fluid mixtures were applied on a cotton swab and then transferred into methanol. A metallic probe was used to sample the methanol solution for subsequent thermal desorption-electrospray ionization mass spectrometry analysis. Altogether, pesticide sampling, transfer, desorption, ionization, and detection took less than 1 min. The reproducibility of this method (n = 6) was shown in the observed low-relative standard deviation (<7%) in the detection of pesticide in oral fluid. The detection limits of the pesticides in oral fluid obtained from four human subjects by thermal desorption-electrospray ionization mass spectrometry were between 1-10 ppb with relative standard deviation 10.7%. Moreover, in this study, linear responses of five pesticides in oral fluid with concentrations between 1 ppb-1 ppm (R2 between 0.9938 and 0.9988) were observed. As the whole analytical process is extremely short, this technique allows for early non-invasive point-of-care identification of pesticides in the oral fluid of self-poisoning patients in the emergency room, providing important toxicological information for decision-making during critical resuscitation.

In vivo positron emission tomography imaging of protease activity by generation of a hydrophobic product from a noninhibitory protease substrate.

PURPOSE: To develop an imaging technology for protease activities in patients that could help in prognosis prediction and in design of personalized, protease-based inhibitors and prodrugs for targeted therapy. EXPERIMENTAL DESIGN: Polyethylene glycol (PEG) was covalently attached to the N-terminus of a hydrophilic peptide substrate (GPLGVR) for matrix metalloproteinase (MMP) to increase hydrophilicity. PEG-peptide was then linked to a hydrophobic tetramethylrhodamine (TMR) domain and labeled with (18)F to form a PEG-peptide-(18)F-TMR probe. Specific cleavage of the probe by MMP2 was tested in vitro by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF). In vivo imaging of MMP2-expressing tumors was evaluated by micro-PET. RESULTS: The hydrophobic TMR fragment (948 Da) was specifically generated by MMP2 enzymes and MMP-expressing HT1080 cells but not control MCF-7 cells. MMP-expressing HT1080 cells and tumors selectively accumulated the hydrolyzed, hydrophobic TMR fragment at sites of protease activity. Importantly, we found that (18)F-labeled probe ((18)F-TMR) preferentially localized in HT1080 tumors but not control MCF-7 tumors as shown by micro-PET. Uptake of the probe in HT1080 tumors was 18.4 ± 1.9-fold greater than in the MCF-7 tumors 30 minutes after injection. These results suggest that the PEG-peptide-(18)F-TMR probe displays high selectivity for imaging MMP activity. CONCLUSIONS: This strategy successfully images MMP expression in vivo and may be extended to other proteases to predict patient prognosis and to design personalized, protease-based inhibitors and prodrug-targeted therapies.

Endocytosis of PEGylated agents enhances cancer imaging and anticancer efficacy.

PEGylated nanoparticles and macromolecules are increasingly used in cancer imaging and anticancer treatment. The role of receptor-mediated endocytosis in the efficacy of these agents, however, has not been clearly defined. Here, we developed a matched pair of endocytic and nonendocytic receptors to directly and unambiguously assess this issue. The ligand-binding domains of the low-density lipoprotein receptor (LDLR) or a truncated LDLR lacking the NPXY endocytosis motif (DeltaLDLR) were replaced with an anti-polyethylene glycol antibody (alphaPEG) to form endocytic alphaPEG-LDLR and nonendocytic alphaPEG-DeltaLDLR receptors. The receptors were stably expressed at similar levels on the surface of HCC36 cells. HCC36/alphaPEG-LDLR cells, but not HCC36/alphaPEG-DeltaLDLR cells, rapidly endocytosed PEG-quantum dots and PEG-liposomal doxorubicin (Lipo-Dox) in vitro and in vivo. Lipo-Dox was significantly more cytotoxic to HCC36/alphaPEG-LDLR cells than to HCC36/alphaPEG-DeltaLDLR cells. HCC36/alphaPEG-LDLR tumors also accumulated significantly more PEGylated near-IR probes (PEG-NIR797) and PEG-liposomal-(111)In than HCC36/alphaPEG-DeltaLDLR tumors in vivo. Furthermore, Lipo-Dox more significantly suppressed the growth of established HCC36/alphaPEG-LDLR tumors as compared with HCC36/alphaPEG-DeltaLDLR tumors. Our data show that endocytosis of PEGylated probes and drugs enhances both cancer imaging and anticancer efficacy, indicating that endocytic receptors are superior targets for the design of cancer imaging probes and immunoliposomal drugs.

Using electrospray-assisted pyrolysis ionization/mass spectrometry for the rapid characterization of trace polar components in crude oil, amber, humic substances, and rubber samples.

We describe the use of electrospray-assisted pyrolysis ionization/mass spectrometry (ESA-Py/MS) to selectively ionize trace polar compounds that coexist with large amounts of nonpolar hydrocarbons in crude oil, amber, humic substances, and rubber samples. Samples of different origins are distinguished rapidly by their positive ion ESA-Py mass spectra without prior separation or chemical pretreatment. During ESA-Py analysis, the samples in their solid or liquid states were pyrolyzed at 590, 630 or 940 degrees C using a commercial Curie-point pyrolysis probe. The gaseous pyrolysates were transferred into a glass reaction cell. The polar compounds (M) in the pyrolysates were then ionized by electrospray ionization (ESI), yielding protonated molecules (MH+). Although the major components of the pyrolysates are nonpolar hydrocarbons, their lack of functional groups that can receive a proton in the ESA-Py source results in no hydrocarbon ion signals being produced; thus, the positive ions detected in ESA-Py mass spectra all result from trace polar components in the pyrolysates.

Characterization of the chemical components on the surface of different solids with electrospray-assisted laser desorption ionization mass spectrometry.

In this study we demonstrate that electrospray-assisted laser desorption ionization (ELDI) mass spectrometry (MS) can be used to rapidly characterize major chemical components on the surfaces of different solids under ambient conditions. The major chemical components in (a) dried milks with different fat contents, (b) different color-regions of a painting, (c) the thin coating on a compact disc, (d) drug tablets, and (e) porcine brain tissue were rapidly characterized as protonated molecules [M+H](+) or sodiated molecules [M+Na](+) by ELDI-MS with minimum sample pretreatment. The ionized ions of synthetic polymer and dye standards were detected directly from dried sample solutions using either positive or negative ion mode. Further structural information for the FD&C Red dye was obtained through tandem mass spectrometric (MS/MS) analysis using an ion trap mass analyzer attached to the ELDI source.

Electrospray ionization from a droplet deposited on a surface-modified glass rod.

This paper reports development of a non-mechanical electrospray ionization (ESI) method to generate electrospray from a droplet deposited on an optical fiber coated with a thin gold or Nafion film. Modification of the surface of the optical fiber in this manner increases its wettability, such that a droplet of the aqueous sample solution can adhere sufficiently strongly to the tip of the fiber. The aqueous sample solution was deposited near the tip of the fiber with a micropipette. When a high voltage (2,000 V) was applied to the fiber by electrical connection through the gold film, the sample solution moved and hung at the tip of the fiber. Simultaneously, ESI was generated from the sample droplet. Multiply charged peptide and protein ions were detected by connecting the ESI source to a quadrupole mass analyzer.