Rethinking prognostic factors in locally advanced or metastatic urothelial carcinoma in the immune checkpoint blockade era: a multicenter retrospective study.

BACKGROUND: Few studies have investigated the safety and efficacy of anti-PD-(L)1 antibodies in metastatic urothelial carcinoma (mUC) in daily clinical practice. Knowledge about the influence of baseline clinical and analytical factors on therapy outcomes is scarce. PATIENTS AND METHODS: We conducted a multicenter retrospective study involving 119 previously treated or untreated mUC patients under anti-PD-(L)1 therapy in a real-world scenario. The objectives of this study were to confirm the safety and efficacy of anti-PD-(L)1 monotherapy and to identify pretreatment factors influencing therapy outcomes. In addition, an independent prognostic model for overall survival (OS) was developed and internally validated. RESULTS: Median OS was 7.8 months [95% confidence interval (CI), 5.4-10.4], median progression-free survival (PFS) was 2.80 months (95% CI, 2.4-3.4), disease control rate (DCR) was 40% (95% CI, 31-49), and overall response rate (ORR) was 24% (95% CI, 15-31). Presence of peritoneal metastases was associated with poor OS [hazard ratio (HR) = 2.40, 95% CI, 1.08-5.33; P = 0.03]. Use of proton-pump inhibitors (PPI) was associated with poor OS (HR = 1.83, 95% CI, 1.11-3.02; P = 0.02) and PFS (HR = 1.94, 95% CI, 1.22-3.09; P = 0.005), and lower DCR (OR = 0.38, 95% CI, 0.17-0.89; P = 0.03) and ORR (OR = 0.18, 95% CI, 0.02-1.60; P = 0.002). The three risk category prognostic model developed included Eastern Cooperative Oncology Group performance status, PPI use, albumin level, presence of liver metastases, and presence of peritoneal metastases variables and was associated with higher risk of death (HR = 3.00, 95% CI, 1.97-4.56; P = 0.0001). CONCLUSIONS: This study confirms anti-PD-(L)1 monotherapy as a safe and effective treatment option in daily clinical practice for mUC patients. It also describes the presence of peritoneal metastases as an independent prognostic factor for OS and underlines the association between PPI use and worse therapeutic outcomes. Finally, it proposes a new easy-to-use risk-assessment model for OS prediction.

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase.

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis-Menten constant (K(M)) of the enzyme and Vmax of the reaction. The obtained K(M) of the reaction was 14 +/- 3 g L(-1) (41 microM). Furthermore, inhibition by adenosine 5'-monophosphate (AMP) was evaluated, and the K(M)(App) value was determined to be 12 +/- 2 g L(-1) (35 microM) for 7.5 and 15 microM AMP, respectively, with Vmax decreasing from 0.1 +/- 0.03 to 0.05 +/- 0.01 g L(-1) min(-1). Therefore, AMP exerted a non-competitive inhibition.

Implementation of terbium-sensitized luminescence in sequential-injection analysis for automatic analysis of orbifloxacin.

Orbifloxacin (ORBI) is a third-generation fluoroquinolone developed exclusively for use in veterinary medicine, mainly in companion animals. This antimicrobial agent has bactericidal activity against numerous gram-negative and gram-positive bacteria. A few chromatographic methods for its analysis have been described in the scientific literature. Here, coupling of sequential-injection analysis and solid-phase spectroscopy is described in order to develop, for the first time, a terbium-sensitized luminescent optosensor for analysis of ORBI. The cationic resin Sephadex-CM C-25 was used as solid support and measurements were made at 275/545 nm. The system had a linear dynamic range of 10-150 ng mL(-1), with a detection limit of 3.3 ng mL(-1) and an R.S.D. below 3% (n = 10). The analyte was satisfactorily determined in veterinary drugs and dog and horse urine.

Assessment of dentifrice adulteration with diethylene glycol by means of ATR-FTIR spectroscopy and chemometrics.

A direct and fast method for determination of the adulterant diethylene glycol (DEG) in toothpaste and gel dentifrices combining attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy with partial least squares (PLS) regression has been proposed. Considering the high heterogeneity of dentifrices available in the market, the possibility of reducing the number of calibration samples for PLS was evaluated. Similar prediction performance was achieved by both employing a large calibration set of 20 dentifrices spiked with different amounts of DEG and a reduced calibration set of seven ones selected by means of hierarchical cluster analysis (HCA). The feasibility of using the simple calibration model to predict DEG adulteration in a wide variety of unknown dentifrice samples increases the applicability of the proposed method. With this approach, DEG was quantified with a root mean squared error of prediction value of 1.1% for a validation set of 40 different dentifrices containing DEG in the range 0-16% (w:w).

Flow-injection solid surface lanthanide-sensitized luminescence sensor for determination of p-aminobenzoic acid.

A single optosensing device based on lanthanide-sensitized luminescence was developed for determination of p-aminobenzoic acid (PABA). The method is based on the formation of a complex between PABA and Tb(III) immobilized on the solid phase (QAE A-25 resin) placed inside the flow cell. NaCl (1 M) was used as carrier solution and HCl (0.05 M) as eluent. The sample solutions of PABA (100 µL) containing Tb(III) and buffered at pH = 6.0 were injected into the carrier stream and the luminescence was measured at λ (ex) = 290 nm and λ (em) = 546 nm. The method shows a linear range from 0.2 to 6.0 µg mL(-1) with an RSD of 1.2% (n = 10) and a sampling frequency of 22 h(-1). A remarkable characteristic of the method is its high selectivity which allows it to be satisfactorily applied to the analysis of PABA in pharmaceutical samples without prior treatment.

Fast determination of salicylic acid in pharmaceuticals by using a terbium-sensitized luminescent SIA optosensor.

In this article, we report the coupling of sequential injection analysis (SIA) and solid phase lanthanide-sensitized luminescence as a detection technique; in this technique, the energy absorbed by the analyte (retained on a solid support) is transferred to the lanthanide ion, which finally emits the luminescence signal. By using this automatic system, the determination of salicylic acid (SA) is easily, rapidly, and selectively achieved. Microspheres of commercial solid support, Sephadex-QAE A-25, are used to fill the flow-through cell and retain analyte and terbium ions; after the signal from SA-terbium complex is obtained, the solid support is easily regenerated by using an ethylenediaminetetraacetic acid disodium salt 2-hydrate (EDTA) solution. Over 200 determinations can be carried out without replacing the resin microbeads. The proposed method shows a 0.045 microg mL(-1) detection limit, with an R.S.D. lower than 3% and a sampling frequency up to 30 samples per hour. The system has been applied to the determination of SA in pharmaceuticals obtained from the Spanish Pharmacopoeia, with satisfactory results; an additional recovery study has been carried out with recoveries close to 100%.

Application of high-performance liquid chromatography-tandem mass spectrometry with a quadrupole/linear ion trap instrument for the analysis of pesticide residues in olive oil.

This article describes the development of an enhanced liquid chromatography-mass spectrometry (LC-MS) method for the analysis of pesticides in olive oil. One hundred pesticides belonging to different classes and that are currently used in agriculture have been included in this method. The LC-MS method was developed using a hybrid quadrupole/linear ion trap (QqQ(LIT)) analyzer. Key features of this technique are the rapid scan acquisition times, high specificity and high sensitivity it enables when the multiple reaction monitoring (MRM) mode or the linear ion-trap operational mode is employed. The application of 5 ms dwell times using a linearly accelerating (LINAC) high-pressure collision cell enabled the analysis of a high number of pesticides, with enough data points acquired for optimal peak definition in MRM operation mode and for satisfactory quantitative determinations to be made. The method quantifies over a linear dynamic range of LOQs (0.03-10 microg kg(-1)) up to 500 microg kg(-1). Matrix effects were evaluated by comparing the slopes of matrix-matched and solvent-based calibration curves. Weak suppression or enhancement of signals was observed (<15% for most-80-of the pesticides). A study to assess the identification criteria based on the MRM ratio was carried out by comparing the variations observed in standard vs matrix (in terms of coefficient of variation, CV%) and within the linear range of concentrations studied. The CV was lower than 15% when the response observed in solvent was compared to that in olive oil. The limit of detection was < or =10 microg kg(-1) for five of the selected pesticides, < or =5 microg kg(-1) for 14, and < or =1 microg kg(-1) for 81 pesticides. For pesticides where additional structural information was necessary for confirmatory purposes-in particular at low concentrations, since the second transition could not be detected-survey scans for enhanced product ion (EPI) and MS3 were developed.

Fluorimetric SIA optosensing in pharmaceutical analysis: determination of paracetamol.

The coupling of sequential injection analysis (SIA) and fluorimetric solid phase transduction is here applied to the determination of paracetamol in pharmaceuticals. The reaction product between the analyte and sodium nitrite in acidic medium is inserted, after alkalinization, in the system. This product is transitorily retained on the active solid sensing phase (the anionic solid support QAE A-25) developing its native fluorescence signal, which is measured at 325/430 nm for the excitation and emission wavelengths respectively. The described system is linear within the range 6.6-80 microg ml(-1), with a 2 microg ml(-1) detection limit and a 2.5% R.S.D (n=10). The proposed fluorimetric SIA optosensor has been applied to the determination of paracetamol in several pharmaceutical preparations, obtaining satisfactory results.

Development of a multicommuted flow-through optosensor for the determination of a ternary pharmaceutical mixture.

The combination of multicommutation and flow-through multioptosensing is presented in this work as a powerful strategy for the routine analysis of active principles in pharmaceuticals. By coupling methodologies, the selectivity and sensitivity of optosensors is maintained, while the use of the multicommutation approach provides additional advantages, such as low reagent consumption, low waste generation and reduced human supervision. The potential of this integration is enhanced when implemented with multiwavelength detection mode. An UV sensor is here developed for the simultaneous determination of three widely used active principles: salicylamide, caffeine and propyphenazone. The measuring wavelengths were 276 nm for caffeine and propyphenazone, and 302 nm for salicylamide. The five three-way solenoid valves used in the system are controlled by Java-written home-made software. The sensor is based on the on-line selective retention of two of the three analytes on a precolumn placed just before the sensing zone and filled with the same solid support than the flow-through cell (C(18) silica gel). This approach allows the sequential arrival of the analytes to the sensing zone, so allowing their determination with only one sample injection. So, the use of C(18) placed, in both the precolumn and the flow-cell combines the advantages of the increase of sensitivity and selectivity in the detection solid zone with the additional increase of the selectivity in the precolumn. The sensor was applied to the determination of the analytes in several pharmaceutical preparation of the Spanish Pharmacopoeia, obtaining satisfactory results.

Development of a photochemically induced fluorescence-based optosensor for the determination of imidacloprid in peppers and environmental waters.

A novel flow-through solid phase spectroscopic assembly implemented with photochemically induced fluorescence (PIF) has been developed for the rapid, sensitive and selective determination of imidacloprid. The pesticide is derivatized on-line by irradiation with ultraviolet light providing an intensively fluorescent compound. The determination is carried out by measuring the fluorescence intensity of the photoproduct once retained on C(18) silica gel filling the flow-cell. The method proposed has been applied to the determination of imidacloprid in natural waters and peppers. The quantification limit (QL) (0.015mgkg(-1)) in peppers was lower than the maximum residue limit (MRL) established by Spanish Legislation (0.5mgkg(-1)). The method also seems to be suitable for environmental water analysis providing satisfactory recoveries (94.0-108.6%). The results obtained in the analysis of real samples are in good agreement with those provided by a reference liquid chromatography (HPLC) method.

Effect of polyethyleneimine ion on the sorption of a reactive dye onto Leacril fabric: electrokinetic properties and surface free energy of the system.

Data are presented on the kinetics, electrokinetics, and surface free energy in the process of adsorption of polyethyleneimine (PEI) as a pretreatment of Leacril, later dyed with the reactive dye Remazol Brilliant Blue R (RBBR). The electrokinetic potential of Leacril is negative, due probably to the presence of sulfonate and sulfate end-group onto Leacril fibers. The zeta potential of Leacril decreases in absolute value as a function of NaCl concentration in solution, probably because of compression of the electrical double layer. The zeta potential of Leacril as a function of the concentration of PEI in solution increases because of the adsorption of PEI ions through chemical reaction between the sulfonate end-groups of Leacril and the amine groups of PEI. The adsorption kinetics shows that an increase in the concentration of PEI, brings about an increase in the amount of RBBR adsorbed onto the fiber. This may be an indication of the chemical reaction between the reactive groups of the polyelectrolyte and dye molecules. The behavior of the surface free energy of the systems involved confirms these conclusions.

Chemiluminescence optosensing implemented with multicommutation: determination of salicylic acid.

In this paper we have coupled, for the first time, chemiluminescent detection with multicommuted optosensing principles. This approach has been implemented with the use of a commercial flow cell of 1mm optical path length filled with an appropriate anionic exchanger gel as chemiluminescence sensing phase. The cell was placed in front of the window of the photosensor module of a home-made luminometer developed in our laboratory and a flat mirror was stuck on the back of the cell. The suitability of using chemiluminescence as detection technique in multicommuted flow-through optosensors has been demonstrated: the determination of salicylic acid by simple oxidation with permanganate on the sensing solid phase was chosen as model reaction. The proposed system allows the determination of salicylic acid in pharmaceuticals, with a sample frequency as high as even 60 samples h(-1) and showing a detection limit of 0.30 microg mL(-1), the linear response range is 1-30 microg mL(-1) and the R.S.D. is 3.1%. Satisfactory results have been obtained when applying the sensor to pharmaceuticals. The accuracy of the proposed methodology has been tested by using a reference method.

Resolution of biparametric mixtures using bead injection spectroscopic flow-through renewable surface sensors.

A new, sensitive and simple bead injection spectroscopy-flow injection analysis (BIS-FIA) sensor with spectrophotometric detection, using a commercially available flow-cell, is described to the determination of biparametric mixtures. As an analytical model, the metallic mixture Cu(II) and Zn(II) has been chosen. The flow-cell (Hellma 138-OS) is filled by injecting in the flow system 300 microl of a homogeneous bead suspension of an anion exchanger gel (Sephadex QAE A-25) previously loaded with the chromogenic reagent 2-carboxyl-2-hydroxy-5-sulfoformazylbenzene (Zincon). A sequential reaction of Cu(II) and Zn(II) with Zincon to form two complexes is performed on the bead sensing support and the absorbance is monitored at 627 nm, after two successive injections from the mixture solution. The sample containing these metal ions is injected into the first carrier (deionized water, pH 5.9), and Cu(II) selectively reacts with Zincon on the beads, developing the analytical signal. Then, 600 microl of 2 M HCl is injected to decompose the complex, and the carrier solution is changed. At pH 11 (second carrier) both Cu(II) and Zn(II) react with the chromogenic reagent, the absorbance now corresponding to both analytes. The eluent is again injected to descompose both complexes. After three analyses the sensing bead surface is not regenerated. Then, beads are automatically discarded from the flow cell by reversal of the flow, and instantaneously transported out of the system. So the procedure exploits the combination of the concepts of flow-through renewable sensors with bead injection spectroscopy. Using a sample volume of 1000 microl, the calibration graph for Cu(II) is linear over the range 0.05 to 1 microg ml(-1) and for Zn(II) from 0.1 to 1.8 microg ml(-1) in the presence of each other. RSDs (%) lower than 5% are obtained for both analytes. The sensor is satisfactorily applied to individual determination or mixture resolution in waters, pharmaceuticals, soils and human hair samples.

Multicommuted flow-through fluorescence optosensor for determination of furosemide and triamterene.

Multicommutation implemented with flow-through optosensors is a very promising area of research. This recent approach benefits from the advantages of both methods and results in high sensitivity, selectivity, and speed, and little waste generation. This paper reports the simultaneous determination of furosemide and triamterene, two widely used diuretics, by measurement of their native fluorescence. The system has been proved to be useful for determination of both analytes in pharmaceutical preparations and for determination of triamterene in human urine and serum. A minicolumn filled with Sephadex SPC-25 microbeads was used to achieve separation of both analytes before detection in a flow-through cell filled with the same resin. The sensor is linear in the range 50-1200 and 0.4-8 ng mL(-1) with detection limits of 15 and 0.1 ng mL(-1) for furosemide and triamterene, respectively.

Solid-phase ultraviolet sensing system for determination of methylxanthines.

In this study the use of a single continuous-flow solid-phase UV spectrophotometric sensing system for determination of methylxanthines was evaluated. Two methods were developed to determine caffeine (CF) and theophylline (TP) in pharmaceuticals and CF and theobromine (TB) in food and beverages. The sensor is based on transient and sequential retention of the analytes on a hydrophobic sensing solid zone (octadecyl silane C18 gel) and detection of their intrinsic UV absorbance. Temporary sequencing of the arrival of the analytes at the sensing zone is achieved by on-line separation of one of the analytes using a pre-column of the same particulate material, placed just before the flow cell. After TB or TP had been carried toward the sensing zone (by the appropriate carrier solution), produced its transitory signal, and been eluted by the carrier, an appropriate eluting solution (25% MeOH) was used to elute CF, which was strongly retained on the minicolumn, so that its transient signal could be recorded. The sensing zone was completely regenerated with this eluting solution, and so was ready for analysis of another sample. After selecting the most suitable conditions, the sensing system was calibrated in the range 1-16 and 1-12 mg L(-1) for CF and TP-TB, respectively, giving detection limits below 0.1 mg L(-1) with RSD values less than 3%. The usefulness of this approach has been evaluated by applying it to the determination of caffeine, theobromine, and theophylline in different samples of food, beverages, and pharmaceutical formulations. The results were in satisfactory agreement with those obtained by use of an HPLC reference method.

A flow-injection renewable surface sensor for the fluorimetric determination of vanadium(V) with Alizarin Red S.

Vanadium(V) is determined by a simple bead injection spectroscopy-flow-injection analysis (BIS-FIA) system with spectrofluorimetric detection using a commercially available flow cell (Hellma 176-QS). The 500mul of a homogeneous bead suspension of an anionic resin (Sephadex QAE A-25) previously loaded with the fluorogenic reagent 1,2-dihydroxyanthraquinone-3-sulfonic acid (Alizarin Red S) was injected to fill the flow cell. Next, V(V) is injected into the carrier and reacts with the immobilized reagent on the active solid support placed in the flow cell to form a fluorescent chelate in the absence of surfactant agents. The complex is so strongly retained on the beads that the regeneration of the solid support becomes extraordinarily difficult, so needing the renovation of the sensing surface which is achieved by means of bead injection. At the end of the analysis, beads are automatically discarded from the flow cell and transported out of the system by reversing the flow. The measurement of fluorescence is continuously performed at an excitation wavelength of 521nm and an emission wavelength of 617nm. Using a low sample volume of 800mul, the analytical signal showed a very good linearity in the range 2-60ngml(-1), with a detection limit of 0.45ngml(-1) and a R.S.D. (%) of 4.22 for 50ngml(-1) of V(V) concentration (n=10). The sensor showed both a good selectivity, which could also be increased by the addition of EDTA and F(-) as masking agents, and applicability to the determination of V(V) in waters, physiological samples (serum and urine) and mussel tissues.

Bead injection spectroscopy-flow injection analysis (BIS-FIA): an interesting tool applicable to pharmaceutical analysis. Determination of promethazine and trifluoperazine.

A bead injection spectroscopy-flow injection analysis (BIS-FIA) system for the spectrophotometric detection of promethazine and trifluoperazine is developed. The sensor is based in the oxidation of the phenothiazines by Fe(III) which is later determined by formation of the complex between Fe(II) and Ferrozine, [FeFz(3)](4-). Immediately, this complex is retained on a homogeneous bead suspension of Sephadex QAE A-25 resin (500 microl) which has been injected previously in the system to fill a commercial flow-cell (Hellma 138-OS). The use of BI with respect to the use of a reusable flow-through sensor is justified because the complex is so strongly retained on the beads that the regeneration of the solid support becomes extraordinarily difficult in the proposed method. At the end of the analysis, beads are automatically discarded from the flow-cell, by reversing the flow, and transported out of the system. The analytical signals are measured at a wavelength of 567 nm, corresponding to the absorbance of the complex. Using a sample volume of 600 microl, the analytical signal showed a very good linearity in the range 0.5-8.0 microgml(-1) and 0.5-10.0 microgml(-1), with detection limits of 0.09 and 0.14 microgml(-1) for promethazine and trifluoperazine, respectively. R.S.D.s (%) lower than 2% were obtained for both analytes. The proposed method is highly selective in the presence of other species that are normally encountered with these analytes. The sensor was satisfactorily applied to pharmaceutical preparations.

Continuous-flow separation and pre-concentration coupled on-line to solid-surface fluorescence spectroscopy for the simultaneous determination of o-phenylphenol and thiabendazole.

A novel and single flow-injection system combined with solid-surface fluorescence detection is proposed in this work for the resolution of a mixture of two widely used pesticides (o-phenylphenol and thiabendazole). The continuous-flow methodology is based on the implementation of on-line pre-concentration and separation of both analytes on the surface of C18 silica gel beads placed just inside the flow cell, implemented with gel-phase fluorimetric multi-wavelength detection (using 305/358 and 250/345 nm as excitation/emission wavelengths for thiabendazole and o-phenylphenol, respectively). The separation of the pesticides was possible owing to the different retention/desorption kinetics of their interactions with the solid support in the zone where the stream impinges on the solid material. No previous separation of the analytes before they reach the flow cell is needed thereby simplifying substantially both the procedure and the manifold. By using a sample volume of 2,600 microL, the system was calibrated in the range 0.5-16 and 5-120 ng mL(-1) with detection limits of 0.09 and 0.60 ng mL(-1) for thiabendazole and o-phenylphenol, respectively. The RSD values (n=10) were about 1% for both analytes. The proposed methodology was applied to environmental water samples and also to various commercial pesticide formulations containing both analytes. Recovery percentages were 97-103% and 98-102% for thiabendazole and o-phenylphenol, respectively.

Multiwavelength fluorescence based optosensor for simultaneous determination of fuberidazole, carbaryl and benomyl.

In the present work, a novel approach is proposed for the simultaneous determination of three widely used pesticides (namely, fuberidazole (FBZ), carbaryl (CBL) and benomyl (BNM)). The proposed method is based on a single continuous-flow solid surface fluorimetric multi-optosensor implemented with the use of a minicolumn placed just before the flow-through cell and filled with C(18) silica gel. The three pesticides are determined from an only injection (simultaneous determination): the minicolumn strongly retains two of them while the third develops a transitory signal when passing through the sensing solid microzone. Then, two alternate eluting solutions appropriately selected perform the sequential elution of the two pesticides from the minicolumn, achieving the detection zone and developing their transitory signals. The proposed optosensor works under optimal sensitivity conditions for all the three analytes because of the use of multi-wavelength fluorescence detection mode, so recording three different signals corresponding at three pairs of optima excitation/emission wavelengths. Using a sample volume of 2100mul, the system was calibrated in the range 0.5-15, 40-800 and 50-1000mugl(-1) with detection limits of 0.09, 6 and 9mugl(-1) for FBZ, CBL and BNM, respectively. The R.S.D values (n=10) were lower than 2% in all cases. The proposed methodology was applied satisfactorily to water samples. Recovery percentages ranging from 97.8 to 101.1%, 97.9 to 103% and from 97 to 105% for FBZ, CBL and BNM, respectively, were obtained.