Quantitation of various indolinyl caged glutamates as their o-phthalaldehyde derivatives by high performance liquid chromatography coupled with tandem spectroscopic detections: derivatization, stoichiometry and stability studies.

Quantification, stability and unique spectroscopic properties of indolinyl-caged glutamates (ICGs), with the o-phthalaldehyde-3-mercaptopropionic acid (OPA-MPA) reagent, were described, at first. As new principle to the field, reactivity and stoichiometry of variously substituted OPA-MPA derivatized ICGs, such as 4-methoxy-7-nitroindolinyl-(MNI-Glu), 4-methoxy-5,7-dinitroindolinyl-(DNI-Glu), 2-dimethylamino-propoxy and dimethylamino-isobutoxy alternatives (2DMA-1PO-DNI-Glu, 1DMA-2P-DNI-Glu and 3DMA-1iBU-DNI-Glu), was demonstrated. Derivatives' stability was determined using high performance liquid chromatography (HPLC) applying simultaneous photodiode array (DAD) and fluorescence (Fl) detections, while their structural identity was confirmed by HPLC-time of flight mass spectrometry (HPLC-TOF-MS). The SH-additive of the reagents was also varied. ICGs react unequivocally, with one OPA-SH-group molecule, in the molar ratios of ([OPA-SH-additive]/[ICG]=1/1, resulting in species with the characteristic isoindole spectral property (EEx/EEm=337/454nm; λmax=337nm). ICGs' isoindole derivatives, due to their sandwich structure, are manifesting the π-π-stacking phenomenon: they fail to show fluorescence. ICGs' stability decreased in the order of MNI-Glu, 2DMA-1PO/1DMA-2PO, 3DMA-1iBU and DNI-Glu, correspondingly, resulting in increasing order of free glutamic acid (GA), as their decomposition product. GA and ICGs were determined as their OPA/MPA derivatives while uncaged species (MNI, DNI and its substituted alternatives) in their initial forms. The practical utility of the method was confirmed analyzing ICGs and their decomposition products, simultaneously. Quantifications' reliability and reproducibility were characterized with the relative standard deviation percentages of responses (RSDs%): for GA 0.41-12 RSD% for ICGs 0.057-7.0 RSD% were obtained. Stability properties of variously substituted, recently introduced ICGs, prepared in laboratories of Institute of Experimental Medicine, were defined.

Systematic derivatization, mass fragmentation and acquisition studies in the analysis of chlorophenols, as their silyl derivatives by gas chromatography-mass spectrometry.

An exhaustive GC-MS sample preparation, derivatization, mass fragmentation and acquisition study was performed, for the simultaneous analysis of chlorophenols (CPs). Selected species were 2-CP, 3-CP, 4-CP, 3,5-dichlorophenol (diCP), 2,5-diCP, 2,6-diCP, 2,4-diCP, 2,3-diCP, 3,4-diCP 2,4,6-trichlorophenol (triCP), 2,4,5-triCP, 2,3,4-triCP, 2,3,4,6-tetrachlorophenol (tetraCP) and pentachlorophenol (pentaCP), in total 14 compounds. As novelties to the field, basic researches, like systematic derivatization, mass fragmentation and acquisition methods have been optimized for the trimethylsilyl (TMS) ether derivatives of CPs. The reactivity of chlorophenols with silylating agents has not been systematically analyzed. Here, we studied the reactivity of 14 chlorophenols with five silylating reagents. The three acquisition techniques, the full scan (FS), the multiple ion monitoring (MIM), and the currently optimized multiple reaction monitoring (MRM) methods, have been compared. We developed a new analytical approach, simultaneously monitoring the fragmentation pattern of the (35)Cl and the (37)Cl containing fragment ions both as precursor and as product ions. This principle resulted in remarkable specificity and sensitivity of detection and quantification; particularly in the cases of the tetraCP and pentaCP derivatives containing the (35)Cl and the (37)Cl fragment ions at an approximate ratio of <1:1. Detailed documentation of the loss of HCl via fragmentation processes, without decomposition of the benzene ring, was attributed to the "ring-walk" mechanism described first for monochlorophenol. Critical evaluation of the derivatization and acquisition protocols was collated and validated with the same characteristics. Data of six point calibration along with the corresponding relative standard deviation percentage (RSD%) values, in the line of FS, MIM and MRM methods (r(2): 0.9987, 0.9992, 0.9989; RSD%: 8.7, 5.6, 8.1), proved to be independent on the acquisition processes. The practical utility of the optimized MRM acquisition techniques was confirmed by the quantitation of the CP contents of Danube River, tap water and distilled water samples. Results confirmed at the first time the primary importance of the MRM acquisition method, even in comparison to the MIM one: we revealed that distilled water contains higher chlorophenol content than tap water, which might have a great significance for the water industry.

The role of the acquisition methods in the analysis of natural and synthetic steroids and cholic acids by gas chromatography-mass spectrometry.

An exhaustive GC-MS acquisition study was performed, for the simultaneous analysis of natural and synthetic steroids and cholic acids (in order to insert them into the last tierce of our multiresidue analysis system), such as androsterone, ß-estradiol, transdehydroandro-sterone, transdehyroandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxyprogesterone-acetate, lithocholic acid, stigmasterol, cholic acid, chenodeoxycholic acid, ß-sitosterol, ursodeoxycholic acid, 3-hydroxy-7-ketocholic acid and dehydrocholic acid, in total 26 compounds. As novelties to the field, for the trimethylsilyl (TMS) oxime ether/ester derivatives of steroids and cholic acids, at first, a tandem mass spectrometric (MS/MS), multiple reaction monitoring (MRM) type acquisition method has been developed in a single run; also for the first time, the three acquisition techniques, the full scan (FS), the selective ion monitoring (SIM), in our case the multiple ion monitoring (MIM) and the currently optimized MRM methods, have been compared; all three, in parallel, under strictly the same derivatization/instrumental conditions, both in matrix free solutions and municipal wastewater from two Hungarian wastewater treatment plants (WWTPs). Critical evaluation of the three acquisition protocols was collated on their analytical performances and validated under the same conditions. The data of six point calibration curves for FS, MIM and MRM methods, showed that both R² (0.9995, 0.9858, 0.9975) and RSD (5.3, 5.8, 5.0), for two parallel derivatizations, each injected three times, proved to be independent of the acquisition processes. Whereas, for the method limit of quantification (LOQ) and the instrument limit of quantification (ILQ) values showed considerable differences. LOQ data, were decreasing in the FS, MIM, MRM line (expressed in ng/L), for all steroids and cholic acids. The same trend was determined in terms of the ILQ values. The practical utility of the optimized acquisition techniques was confirmed by the quantitation of the steroids and cholic acids contents of wastewater samples. Results confirmed the importance of the MRM acquisition method, even in comparison to the MIM one: with particular interest in selected cases: avoiding the extreme overestimation of the ß-estradiol (156-1325%) and that of the ethinylestradiol (582-831%) concentrations in the wastewater samples.

The role of the acquisition methods in the analysis of the non-steroidal anti-inflammatory drugs in Danube River by gas chromatography-mass spectrometry.

In this paper authors describe a GC-MS acquisition study, relating to the most common, non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketoprofen and diclofenac. As novelties to the field, for the trimethylsilyl (TMS) oxime ester derivatives of NSAIDs, at first, a tandem mass spectrometric (MS/MS) acquisition method has been developed, and, also for the first time, the three acquisition techniques, the full scan (FS), the selective ion monitoring (SIM) and the currently optimized MS/MS ones, have been compared: all three in parallel, under strictly the same derivatization/instrumental conditions, both from model solutions and from the Danube River samples. Critical evaluation of the three acquisition protocols was collated on their analytical performances and validated with the same characteristics like the six point calibration curve, the relative standard deviation percentages (RSD%) of parallel tests, the limit of quantitation (LOQ) and the instrumental limit of quantitation (ILQ) values. Data of six point calibration (r(2)>or=0.997) and RSD% (average: 5.8 RSD%) values proved to be independent on the acquisition methods, while, LOQ and ILQ values furnished considerable differences. Decreasing LOQ data, (expressed in ng/L concentrations) were obtained in the FS, SIM, MS/MS line for ibuprofen (1.0, 0.43, 0.41), naproxen (1.1, 1.0, 0.42), ketoprofen (2.6, 1.0, 0.49) and diclofenac (1.4, 0.41, 0.21), respectively. The same trend was determined in terms of the ILQ values. The practical utility of the optimized MS/MS technique was confirmed by the quantitation of the NSAID contents of the Danube River samples, determined by all three acquisition techniques. Results obtained confirmed the primary importance of the MS/MS acquisition method, even in comparison to the SIM one: avoiding the extreme overestimation of the ibuprofen (approximately 100%) and ketoprofen (approximately 400%) concentrations in the Danube River samples.

Multiresidue analysis of pollutants as their trimethylsilyl derivatives, by gas chromatography-mass spectrometry.

This paper reports a multiresidue analysis procedure which permits the identification and quantification of sixty-three water-soluble pollutants. Subsequent to their solid-phase extraction (SPE) enrichment, analyses of species have been carried out from one solution, by a single injection, as their trimethylsilyl-oxime ether/ester derivatives, by gas chromatography-mass spectrometry, within 31min. Based on our optimized extraction, derivatization and mass fragmentation studies separation have been performed in the total ion current mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Including various pharmaceuticals, benzoic acid, its substituted species, different aromatic carboxylic acids, cholic acids, unsaturated and saturated fatty acids, aliphatic dicarboxylic acids, as well as synthetic pollutants of various origins (2,4-di-tert-butylphenol, different phthalates). Standard compounds were added to 500 mL effluent wastewater samples, at three concentrations (1-5 microg/L, 5-10 microg/L and 10-20 microg/L). Recoveries, using the Waters Oasis cartridges performing extractions at pH 2, pH 4 and pH 7 proved to be the optimum at pH 4 (average recoveries (94.5%), except for cholesterol (10%), paracetamol (18%) and 2,5-dihydroxybenzoic acid (25%). Carbamazepine could be recovered at pH 7, only. Responses, obtained with derivatized standards proved to be linear in the range of 4-80 microg/L levels. Limit of quantitation values varied between 0.92 ng/L (4-hydroxyphenylacetic acid) and 600 ng/L (dehydrocholic acid) concentrations. One of the most important messages of this work is the confirmation of the origin of blank values. It was shown that contaminants, mainly 2,4-di-tert-butylphenol, different phthalates and fatty acids, are sourced both from the reagents and mainly from the SPE procedure, independent on the cartridge applied. Reproducibilities, characterized with the relative standard deviations (RSDs) of measurements, varied between 0.71% and 10%, with an average of 4.38% RSD. The practical utility of the method was shown by the identification and quantification of the pollutant contents of Hungarian influent and effluent wastewaters (for six consecutive months and that of the Danube River for 2 months).

Gas chromatography-mass spectrometry of the trimethylsilyl (oxime) ether/ester derivatives of cholic acids: their presence in the aquatic environment.

This paper presents a derivatization, mass fragmentation study relating to the most common six cholic acids, such as cholic, lithocholic, chenodeoxycholic, ursodeoxycholic, 3-hydroxy,7-ketocholanic and dehydrocholic acids, identified and quantified as pollutants in the aquatic environment at the first time. Derivatizations have been performed with the two-step process (1: oximation, 2: silylation) varying the time and temperature of both reactions. Optimum responses have been obtained after 30 min oximation with hydroxylamine.HCl and 90 min silylation with hexamethyldisilazane and trifluoroacetic acid at 70 degrees C. Fragmentation patterns of the trimethylsilyl (oxime) ether/ester derivatives of all six cholic acids provided the theoretically expected, fully derivatized compounds. Reproducibility/linearity of derivatives calculated on the basis of the corresponding selective fragment ions, characterized by the relative standard deviation percentages of measurements, proved to be < or =4.9 (RSD%). The practical utility of the method was shown by the identification and quantification of cholic acids as pollutants in the aquatic environment. Subsequently to a solid phase extraction study varying the pH of extractions (pH 2, pH 4 and pH 7), applying the OASIS cartridges, it has been confirmed that the recoveries for all six cholic acids are acceptable, varying between 77% and 104%, and are independent on the pH. The total cholic acid content of a Hungarian wastewater plants' influent wastewater varied between 184 microg/L and 356 microg/L, while the Danube rivers' cholic acid content was 4.1 microg/L, only.

Identification and quantification of ibuprofen, naproxen, ketoprofen and diclofenac present in waste-waters, as their trimethylsilyl derivatives, by gas chromatography mass spectrometry.

This paper reports a derivatization, mass fragmentation study relating to the most common, non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, ketoprofen and diclofenac, identified and quantified in the aquatic environment. Derivatizations have been performed with four silylation reagents in order to select the most proper one, taking into account analytical and financial points of view, equally. The tested reagents were N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide (MTBSTFA) and for this purpose at the first time, hexamethyldisilazan (HMDS)+trifluoroacetic acid (TFAA). Varying derivatization time and temperature, taking into consideration chemical and financial advantages, HMDS+TFAA proved to be the optimum selection. Responses of derivatives have been compared, as a function of the ionization technique (external/internal ionization), as well as on the treatment of compounds' selective fragment ions (SFIs): (i) extracting the corresponding, characteristic m/z masses from TIC elutions and (ii) from SIM elutions, in parallel. Reproducibilities of measurements, expressed in relative standard deviation percentages (R.S.D.%), including the nanogram and the low picogram levels of injected derivatives, provided an average between 0.93 R.S.D.% and 4.11 R.S.D.%. NSAIDs' enrichment was performed with solid-phase extraction (SPE), applying the Oasis HLB (Waters) cartridges: recoveries in the 1-6 microg L(-1) range varied between 84% and 111%, with an average reproducibility of 6.4 R.S.D.%. The utility of the optimized derivatization method is presented, on monthly basis, by the identification and quantitation of the ibuprofen, naproxen, ketoprofen and diclofenac content of the influent and effluent waste-water samples obtained from a Hungarian waste-water treatment plant.