Development of a new quantification method for organic acids in urine as potential biomarkers for respiratory illness.

Asthma and chronic obstructive pulmonary disease (COPD) are common respiratory disorders that have similar clinical presentation and misdiagnosis may lead to improper treatment. There is a need for a better, non-invasive test for the differentiation of asthma and COPD. In this study, we developed a new validated LC-MS/MS method for 17 urinary organic acids that could serve as potential biomarkers. Human urine samples were collected from adults with asthma or COPD. LC-MS/MS was performed using the differential isotope labeling approach. 4-(Dimethylamino) phenacyl bromide (DmPA) was used for derivatization using two different carbon isotopes, allowing for the formation of internal standard for each metabolite. Gradient elution was employed on a C18 column while the LC-MS/MS operated in the multiple reaction monitoring mode (MRM). Regulatory guidelines were used for method validation. Partial Least Squares Discriminative Analysis (PLS-DA) was applied to the log-transformed values of metabolites in each group of asthma and COPD subjects. Full validation in targeted metabolomics is scarce with usually limited number of metabolites, unlike fit-for-purpose approach. Due to the endogenous nature of the metabolites, numerous challenges were encountered during method development and validation, such as the lactic acid interference from the surrounding environment. The required specificity, accuracy and precision was successfully achieved. The method was fully validated, ensuring robustness and reproducibility when analyzing patient samples. The method was applied to analyze human urine samples and PLS-DA analysis showed differentiation of asthma and COPD subjects (R2 0.89, Q2 0.68). As targeted metabolomics is expanding to the clinical sphere, more validated methods/strategies are needed. Our work will expand the current knowledge-base regarding targeted metabolomics.

Tandem mass spectrometric analysis of novel peptide-modified gemini surfactants used as gene delivery vectors.

Diquaternary ammonium gemini surfactants have emerged as effective gene delivery vectors. A novel series of 11 peptide-modified compounds was synthesized, showing promising results in delivering genetic materials. The purpose of this work is to elucidate the tandem mass spectrometric (MS/MS) dissociation behavior of these novel molecules establishing a generalized MS/MS fingerprint. Exact mass measurements were achieved using a hybrid quadrupole orthogonal time-of-flight mass spectrometer, and a multi-stage MS/MS analysis was conducted using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were operated in the positive ionization mode and are equipped with electrospray ionization. Abundant triply charged [M+H]3+ species were observed in the single-stage analysis of all the evaluated compounds with mass accuracies of less than 8 ppm in mass error. MS/MS analysis showed that the evaluated gemini surfactants exhibited peptide-related dissociation characteristics because of the presence of amino acids within the compounds' spacer region. In particular, diagnostic product ions were originated from the neutral loss of ammonia from the amino acids' side chain resulting in the formation of pipecolic acid at the N-terminus part of the gemini surfactants. In addition, a charge-directed amide bond cleavage was initiated by the amino acids' side chain producing a protonated α-amino-ε-caprolactam ion and its complimentary C-terminus ion that contains quaternary amines. MS/MS and MS3 analysis revealed common fragmentation behavior among all tested compounds, resulting in the production of a universal MS/MS fragmentation pathway. Copyright © 2017 John Wiley & Sons, Ltd.

Establishment of tandem mass spectrometric fingerprint of novel antineoplastic curcumin analogues using electrospray ionization.

RATIONALE: Curcumin analogues are antineoplastic agents, designed based on the structure of the spice turmeric with structural modifications aiming at enhancing potency. The goal is to identify the common tandem mass spectrometric (MS/MS) behavior of 13 novel curcumin analogues. Such knowledge is critical for their biological assessment, including metabolite identification and pharmacokinetic evaluation. METHODS: Both detection of the protonated molecules [M + H](+) of the synthesized compounds and determination of their exact molecular masses were achieved with hybrid quadrupole orthogonal time-of-flight mass spectrometry (QqTOF-MS). Low-energy collision-induced dissociation (CID)-MS/MS analysis was performed using triple quadrupole linear ion trap mass spectrometry (QqLIT-MS). Both instruments were equipped with an electrospray ionization (ESI) source. MS(3) and neutral loss experiments were performed using QqLIT-MS to confirm the genesis of the observed product ions. RESULTS: Abundant [M + H](+) molecules were formed using the QqTOF-MS hybrid instrument with mass accuracies below 6 ppm. CID-MS/MS dissociation studies were centered on the piperidone ring of curcumin analogues; twelve common product ions have been identified from the fission of the various bonds within the piperidone moiety. There was a tendency for the formation of highly conjugated product ions, stabilized via resonance. The variety of the side-chain substituents at the nitrogen atom resulted in side-chain-specific product ions. CONCLUSIONS: The ESI-CID-MS/MS analysis of curcumin analogues revealed a common fragmentation behavior of all tested compounds, which gave diagnostic product ions identified for each molecule. The established MS/MS behavior will be applied to determine metabolic by-products of curcumin analogues as well as to develop targeted identification/quantification methods within biological extracts.

The unexpected formation of [M - H]+ species during MALDI and dopant-free APPI MS analysis of novel antineoplastic curcumin analogues.

Unusual ionization behavior was observed with novel antineoplastic curcumin analogues during the positive ion mode of matrix-assisted laser desorption ionization (MALDI) and dopant-free atmospheric pressure photoionization (APPI). The tested compounds produced an unusual significant peak designated as [M - H](+) ion along with the expected [M + H](+) species. In contrast, electrospray ionization, atmospheric pressure chemical ionization and the dopant-mediated APPI (dopant-APPI) showed only the expected [M + H](+) peak. The [M - H](+) ion was detected with all evaluated curcumin analogues including phosphoramidates, secondary amines, amides and mixed amines/amides. Our experiments revealed that photon energy triggers the ionization of the curcumin analogues even in the absence of any ionization enhancer such as matrix, solvent or dopant. The possible mechanisms for the formation of both [M - H](+) and [M + H](+) ions are discussed in this paper. In particular, three proposed mechanisms for the formation of [M - H](+) were evaluated. The first mechanism involves the loss of H2 from the protonated [M + H](+) species. The other two mechanisms include hydrogen transfer from the analyte radical cation or hydride abstraction from the neutral analyte molecule.