On-line Hydrolysis Research Articles

Hands free urine drug confirmations with online hydrolysis

Introduction: Urine drugs of abuse (DOA) screening results require confirmation by suitable mass spectrometry methods. Drug panel confirmations are often grouped by class and can involve a variety of sample preparation techniques including liquid-liquid or solid phase extraction and a hydrolysis step to liberate target drugs from their glucuronide bonds. An ‘all-in-one’ analytical method incorporating urine hydrolysis, minimal sample preparation and multiple drug class confirmations was desired.

Mechanism of improving hydrogenation of Mg by in-situ formation of Al* in hydriding combustion synthesis

Magnesium hydride (MgH2) is an ideal solid hydrogen storage material and on-line hydrolysis hydrogen source. However, the synthesis of MgH2 usually requires harsh conditions of high temperature, high pressure and long duration, bringing great challenges to production cost and safety. In this paper, MgH2 is prepared by hydriding combustion synthesis (HCS) and the mechanism of improving Mg hydrogenation properties by the in-situ formation of Al* is revealed. Under low hydrogen pressure of 1 MPa, the hydrogenation degree of Mg can be greatly increased from 9.6% of pure Mg to 90.5% with 10 mol% Al added. In the activation stage, Al reacts with Mg to form Mg17Al12, which brings rich grain boundaries and provides rapid diffusion channels for hydrogen. Subsequently, in the hydrogenation stage, Mg17Al12 reacts with H2 to generate Al* in-situ, which are evenly distributed and act as nucleation sites for MgH2. Hydrogen molecules diffuse rapidly along the interface between Mg/MgH2 and Al* and are easily adsorbed and dissociated on the surface of nano-scale Al* . The unique disproportionation hydrogenation reaction of in-situ forming highly active metallic substances as excellent catalysts provides a new idea for the modification of Mg-based hydrogen storage materials.

Development and characterization of mitochondrial membrane affinity chromatography columns derived from skeletal muscle and platelets for the study of mitochondrial transmembrane proteins.

Mitochondrial membrane fragments from human platelets and monkey skeletal muscles were successfully immobilized onto immobilized artificial membrane chromatographic support for the first time, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. These columns were validated by characterization of translocator protein (TSPO), where multiple concentrations of dipyridamole were run and the binding affinities (Kd) determined. Further, the relative ranking data of TSPO ligands was consistent with previously reported rankings for both, the platelet (MMAC-Platelet) and the skeletal muscle (MMAC-Muscle) column (dipyridamole>PK11195>protoporphyrin IX>rotenone). The functional immobilization of the F-ATPase/ATP synthase was demonstrated on MMAC-Muscle column. Online hydrolysis of ATP to ADP and synthesis of ATP from ADP were both demonstrated on the MMAC-Muscle column. Hydrolysis of ATP to ADP was inhibited by oligomycin A with an IC50 of 40.2±13.5nM (∼60% reduction in ATP hydrolysis, p<0.001), similar to previously reported values. Additionally, the Michaelis-Menten constant (Km) for ADP was found to be 1525±461μM based on the on column dose-dependent increase in ATP production.

Multifunctional Hydrogels with Reversible 3D Ordered Macroporous Structures.

Three-dimensionally ordered macroporous (3DOM) hydrogels prepared by colloidal crystals templating display highly reversible shape memory properties, as confirmed by indirect electron microscopy imaging of their inverse replicas and direct nanoscale resolution X-ray microscopy imaging of the hydrated hydrogels. Modifications of functional groups in the 3DOM hydrogels result in various materials with programmed properties for a wide range of applications.

Immobilization of thermostable β-glucosidase variants on acrylic supports for biocatalytic processes in hot water

Two variants of the thermostable β-glucosidase TnBgl1A (wt and N221S/P342L) from Thermotoga neapolitana were immobilized on acrylic supports (Eupergit® C, Eupergit® C250L, and cryogel) and evaluated at conditions close to the boiling point of water. Thermo-gravimetric analysis showed the supports to be stable <250 °C. Both wt and N221S/P342L were covalently bound to oxirane-groups respectively via glutaraldehyde spacers, and for coupling reactions 26 Lys and 20 Ser/Thr were surface-located. Immobilized enzymes were active on all supports in the temperature range 80–95 °C, but the observed specific activity was low (≤19 U mg−1) using the cryogel. More than 91% of the initial activity was maintained after ten times recycling, and the same was recovered after 3 months storage at 4 °C for Eupergit® supports by simply incubating the preparation with bovine serum albumin. No storage loss was detectable on cryogels. The glutaraldehyde spacer improved activity on cryogels, but not on Eupergit® supports. Immobilization on Eupergit® C250L yielded the highest observed specific activity (254 U mg−1 for N221S/P342L) in a procedure including blocking of free oxirane-groups by BSA. This biocatalyst was used for on-line hydrolysis of quercetin-glucosides in a yellow onion extract at 80 °C, proving the immobilized biocatalyst to be promising in on-line systems for extraction and hydrolysis using hot pressurized water.

In-Capillary Screening of Matrix Metalloproteinase Inhibitors by Electrophoretically Mediated Microanalysis with Fluorescence Detection

A capillary electrophoresis-based method with enzymatic reaction inside the capillary for the screening of matrix metalloproteinase (MMP) inhibitors has been developed. MMP-2 and MMP-9, which have been considered as promising targets for cancer therapy, were selected as the model enzymes. The hydrolysis of a fluorogenic substrate catalyzed by MMPs was determined by measuring the increase in fluorescence. For high-throughput screening, the short-end injection was employed. The enzyme, substrate containing inhibitors, and enzyme solutions were injected from the outlet of the capillary via the sandwich mode. They were mixed by alternating the potential at positive and negative polarities. Online hydrolysis, separation, and detection were achieved in 70 s with approximately 0.87 fmol of MMP required for each assay. The applicability of electrophoretically mediated microanalysis (EMMA) with fluorescence detection to estimate the inhibitory mechanism and to determine the IC(50) values was evaluated for two natural inhibitors, epigallocatechin gallate and oleic acid. A few other natural compounds such as resveratrol, quercetin, caffeic acid, glucosamine, and doxycycline were also screened to test their inhibitory potency. The results obtained were compared with those obtained by offline enzyme assay and confirm the effectiveness of the present method. A rapid, cost-effective, and fully automated method for MMP inhibitor screening is proposed.

Flow injection spectrophotometric determination of methamidophos using online hydrolysis

A new protocol for the online spectrophotometric determination of methamidophos has been developed. The method is based on online hydrolysis of methamidophos, and the resulting hydrolyzed product is reacted with sodium nitroproside to form a colored complex. The calibration curve is linear over the concentration range of 0.5-20 microgml(-1), with a molar absorptivity of 2.5x10(4) L mol(-1) cm(-1). The method is fast and reproducible with a sample throughput of 90 samples/h. The method is successfully applied to formulations and real samples.

Fast and repetitive in-capillary production of [18F]FDG

The increasing demand for radiopharmaceuticals to be provided reproducibly and flexibly with high frequency for clinical application and animal imaging would be better met by improved or even new strategies for automated tracer production. Radiosynthesis in microfluidic systems, i.e. narrow tubing with a diameter of approximately 50-500 microm, holds promise for providing the means for repetitive multidose and multitracer production. In this study, the performance of a conceptually simple microfluidic device integrated into a fully automated synthesis procedure for in-capillary radiosynthesis (ICR) of clinical grade [(18)F]FDG was evaluated. The instrumental set-up consisted of pumps for reagent and solvent delivery into small mixing chambers, micro-fluidic capillaries, in-process radioactivity monitoring, solid-phase extraction and on-column deprotection of the (18)F-labelled intermediate followed by on-line formulation of [(18)F]FDG. In-capillary(18)F-fluorination of 2.1 micromol 1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulphonyl-beta-D-mannopyranose (TATM; precursor for [(18)F]FDG) in acetonitrile (MeCN) at a flow rate of 0.3 ml/min within 40 s and subsequent on-line hydrolysis of the intermediate by treatment with 0.3 M NaOH for 1 min at 40 degrees C resulted in a radiochemical yield of 88 +/- 4% within <7 min. Reproducibility, robustness and suitability as a fast and efficient radiopharmaceutical research tool for (18)F-fluorination was demonstrated by eight independent, sequentially performed ICRs which provided identical tracer quality (radiochemical purity >97%, MeCN <5 microg/ml) and similar absolute yields (approximately 1.4 GBq). The described ICR process is a simple and efficient alternative to classic radiotracer production systems and provides a comparatively cheap instrumental methodology for the repetitive production of [(18)F]FDG with remarkably high efficiency and high yield under fully automated conditions. Although the results concerning the levels of activity need to be confirmed after installation of the equipment in a suitable GMP hot-cell environment, we expect the instrumental design to allow up-scaling without major difficulties or fundamental restrictions. Furthermore, we are convinced that similar or nearly identical procedures, and thus instrumentation, will allow ICR of other (18)F-labelled radiopharmaceuticals.

Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives

Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (μIMER) made of porous silicon with the purpose of investigating the use of such μIMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the μIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the μIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The μIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing μIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the μIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight.

FLOW INJECTION SPECTROPHOTOMETRIC DETERMINATION OF PROPOXUR WITH DIAZOTIZED 4-AMINOBENZOIC ACID

ABSTRACT A flow injection spectrophotometric method for the determination of propoxur with diazotized 4-aminobenzoic acid as the coupling agent was developed. The determination involves on-line hydrolysis at room temperature of the propoxur with sodium hydroxide. The resulting 2-isopropoxyphenol is coupled with diazotized 4-aminobenzoic acid in order to achieve an appropriate selectivity and sensitivity to the spectrophotometric measurements. The calibration curve is linear over the range 1–10 mg L−1 of propoxur. The proposed method has provided a detection limit of 0.15 mg L−1 of propoxur and a sample frequency of 120 injections per hour. The relative standard deviation of six independent determinations of a sample containing 5 mg L−1 propoxur was 0.74%. The suitability of the proposed procedure for the determination of propoxur in commercial pesticide formulations was studied. The procedure provides results comparable to those obtained by the liquid chromatography analysis.

Rapid spectrophotometric determination of fosfestrol following on-line hydrolysis by alkaline phosphatase using flow injection and chasing zones

A new flow injection (FI) method is reported for the spectrophotometric determination of fosfestrol (diethyl-stilbestrol (DES) diphosphate) in pharmaceutical formulations. The proposed method is based on the on-line hydrolysis of the analyte by alkaline phosphatase (Al-Pase) using a “chasing zones” FI manifold. The orthophosphate ions, thus, generated are determined spectrophotometrically ( λ max=690 nm) using the molybdenum blue approach. The chemical and FI variables affecting the enzymatic reaction were investigated. The proposed method is very precise ( s r=1.1% at 1×10 −4 mol l −1 fosfestrol, n=12), fast (allowing up to 40 samples h −1 to be analyzed) and has a determination range of 2×10 −5 to 2×10 −4 mol l −1, with a satisfactory 3 σ detection limit of 5×10 −6 mol l −1. The method was shown to provide accurate determinations of the fosfestrol concentration in a pharmaceutical formulation, giving relative errors, e r, of +0.6 and −0.5% compared to the value stated by the supplier (Asta Medica Inc.) and the concentration derived using a method recommended by the United States Pharmacopoeia XXI, respectively. In addition, the average recoveries of known amounts of the analyte ranged between 99.2 and 101.2%.

Determination of humic acid in natural waters by flow injection analysis with chemiluminescence detection

A fast and sensitive direct chemiluminescence flow injection procedure for determination of humic acid in natural waters was developed. N-bromosuccinimide (NBS) after on-line hydrolysis was used as a reagent evolving chemiluminescence by the oxidation of humic acid. Use of glycine as a sensitizer enhances the signal magnitude about 100 times. The detection limit is 0.012 mg l −1 and 180 samples per hour can be determined. Almost all substances occurring in typical fresh water at normal level do not disturb the determination. Only phenols with one hydroxyl group are serious interferents, which were removed by evaporation.

Potentiometric determination of acetylsalicylic acid by sequential injection analysis (SIA) using a tubular salicylate-selective electrode.

This paper deals with the development of an automated procedure for formulation assays and dissolution tests based on a sequential injection analysis (SIA) system involving an ion-selective electrode as sensing device. Construction of a tubular salicylate (Sal) selective electrode suitable for potentiometric determination of acetylsalicylic acid (Asa) in pharmaceutical formulations is described. The flow-through electrode is formed by a PVC membrane containing 29.2% (w/w) PVC, 5.8% (w/w) tetraoctylammonium salicylate (ionic sensor), 58.5% o-nitrophenyloctylether (plasticizer) and 6.5% (w/w) p-tert-octylphenol (stabilising additive which increases electrode selectivity). The calibration range is 0.05--10 mM Sal, the limit of detection (LOD) is 0.05 mM Sal, the slope is 56.0 mV per decade at 22 degrees C. The R.S.D. is 0.20% (15 readings) when determining 2.5 mM Sal in standard solution. The electrode is used for sensing Asa after its on-line chemical hydrolysis to Sal in a SIA system. The sampling rate is 6 h(-1) but for the dissolution tests the frequency is increased to 20 h(-1). The SIA set-up is employed for the assay of Asa in plain tablets, composed tablets and effervescent tablets and for performing dissolution tests of normal and sustained release tablets. Results obtained by this technique compare well with those required by the US Pharmacopoeia XXIV.

Selective determination of orthophosphate and total inorganic phosphates in detergents by flow injection photometric method

A flow injection photometric system was developed for the determination of orthophosphate and total inorganic phosphates in detergents. While orthophosphate was directly determined in the presence of other phosphates by utilizing the kinetic discrimination of flow injection analysis, total inorganic phosphates was analyzed after on-line hydrolysis of polyphosphates in 2.5 mol l −1 sulfuric acid for 50 s under 145°C. Sodium dodecyl sulfate (SDS) was added to mask the interference of non-ionic surfactants. The detection limits and the sampling rates were 2.5 mg l −1 P 2O 5 and 40 h −1 for total inorganic phosphates, and 1.0 mg l −1 P 2O 5 and 80 h −1 for orthophosphate determination. The proposed method was applied to analyze orthophosphate and total inorganic phosphates in washing powders. The experimental results are in good agreement with those obtained by the Chinese national standard methods.

Determination of carbofuran in pesticide formulations by flow injection spectrophotometry

A flow injection spectrophotometric method for the determination of carbofuran in commercial pesticide formulations was developed. The determination involves on-line hydrolysis of the extracted carbofuran at room temperature with sodium hydroxide. The resulting carbofuran-phenol is coupled with diazotized 4-aminobenzoic acid in order to achieve an appropriate selectivity and sensitivity for the spectrophotometric measurements. The calibration curve is linear over the range 1–10 mg litre−1 of carbofuran. The proposed method has a detection limit of 0.15 mg litre−1 of carbofuran and a sample frequency of 120 injections per hour. The relative standard deviation of six independent determinations of a sample containing 1 mg litre−1 carbofuran was 0.6%. The suitability of the proposed procedure for the determination of carbofuran in commercial pesticide formulations was studied. The procedure provides results comparable to those obtained by liquid chromatographic analysis. © 2000 Society of Chemical Industry

Determination of acetylsalicylic acid by FIA-potentiometric system in drugs after on-line hydrolysis

A potentiometric flow injection (FI) system was developed for the acetylsalicylic acid (ASA) determination in drugs, without previous treatment. The tubular potentiometric electrode for salicylate (SA) was based on tricaprylyl-trimethyl-ammonium-salicylate (aliquat-salicylate) as the ion-exchanger, supported on poly(ethylene- co-vinyl-acetate) (EVA) matrix and applied directly onto a conducting support. The standards and samples were freshly prepared in ethanol solution (0.10 mol l −1 Tris–SO 4 buffer, pH 8.0, containing 0.25 mol l −1 Na 2SO 4 and 8.0% v/v ethanol) to facilitate the dissolution of ASA and were injected directly into the system. The SA formed due to the on-line alkaline hydrolysis of alcoholic ASA solution, with 0.50 mol l −1 NaOH (coil, 50 cm length), was monitored by the tubular electrode after neutralization with 0.25 mol l −1 H 2SO 4. A solution of 0.10 mol l −1 Tris–SO 4 buffer (pH 8.0), containing 0.25 mol l −1 Na 2SO 4 was employed as carrier. In optimized conditions (flow rate of 2.1 ml min −1 and volume of injection of 150 μl), the tubular electrode showed a linear response to ASA in the concentration range between 4.0×10 −3 and 4.0×10 −2 mol l −1. A conversion factor of ASA to SA of 85% occurs in these conditions with an increase of about 130% in the signal to the system with on-line hydrolysis (three-channel) in comparison to the system without (one-channel). The response time of the electrode was about 5 s with an analytical frequency of 28 samples per h and a relative standard deviation (R.S.D.) of 2.1% for 30 successive injections. Determinations of ASA in tablet samples by the proposed method exhibited relative differences of 1.0–3.5%, compared to the official method of the British Pharmacopoeia. The useful lifetime of the sensor was greater than 1 month, in continuous use.

A clean analytical method for the spectrophotometric determination of formetanate incorporating an on-line microwave assisted hydrolysis step

A fast and completely automated procedure is proposed for the spectrophotometric determination of formetanate in waters by means of its reaction with p-aminophenol (PAP). The method involves the on-line alkaline hydrolysis of formetanate to m-aminophenol (MAP) and its reaction, in the presence of KIO 4 as oxidant agent, with the quinoneimine form of PAP, to form a blue indophenol dye which absorbs at 576 nm. The on-line hydrolysis can be carried out in a 6 m reaction coil located inside the cavity of a domestic microwave oven operated at 650 W, or in a 4 m reaction coil located inside the cavity of a Microdigest 301 microwave system operated at 60 W and allows us, in both cases, a complete hydrolysis of formetanate thus improving the complete automation of the analytical procedure. After the measurement step, the analytical waste is merged with a TiO 2 slurry and then detoxified by on-line UV-irradiation.

Fluorometric Determination of Phospholipids by High-Performance Liquid Chromatography with Postcolumn On-Line Hydrolysis under UV-Irradiation, Phosphomolybdic Acid and Thiamine–Thiochrome Reactions

A postcolumn phosphate-selective fluorescence detection system was developed for the determination of phospholipids by high-performance liquid chromatography. Phospholipids were separated on a silica gel column and then on-line hydrolyzed by photolysis in the presence of titanium dioxide under UV-irradiation (253.7 nm). The resultant orthophosphate was reacted with a molybdenum reagent to form phosphomolybdic acid, which was then reacted with the alkaline thiamine reagent. Finally, the fluorescence intensity of the resultant thiochrome converted from thiamine by phospho-molybdic acid was measured by setting the excitation wavelength at 380 nm and the emission wavelength at 442 nm. The calibration curves for phospholipids under the gradient elution program were linear (correlation coefficient, 0.984–0.991) in the range 0.02–0.5 mg ml-1 (injection volume, 10 m l). When every 2 μg each of five phospholipids was repeatedly injected into this system, the relative standard deviations were between 4.14 and 4.76% (n=5).

CEDIA dau Benzodiazepine screening assay: a reformulation.

The CEDIA dau Benzodiazepine assay has been reformulated to include online hydrolysis of urinary benzodiazepine glucuronide conjugates. The new antibody possesses enhanced cross-reactivities toward the low-dose benzodiazepines, which are excreted at low urinary drug-metabolite concentrations. The screening method was evaluated using lorazepam as the probe benzodiazepine. Four subjects each consumed a 1-mg lorazepam tablet. Sequential urine voids over the same time intervals were collected for the next 48 h. Twelve postdose urine samples were collected from each subject. Positive results were obtained from 5-24 h to 2-35 h using a 200-ng/mL nitrazepam calibration cutoff. There was no practical difference between hydrolyzing online with the supplied E. coli beta-glucuronidase or offline with Helix pomatia beta-glucuronidase purchased separately. Without hydrolysis, all urine samples tested negative. The cross-reactivities of lorazepam in terms of nitrazepam calibration equivalents, varied from 108 to 178% for lorazepam concentrations between 50 and 2500 ng/mL. Lorazepam glucuronide gave cross-reactivities (expressed as lorazepam base) between 72 and 136% using the online hydrolysis procedure with E. coli beta-glucuronidase. Offline hydrolysis with Helix pomatia gave cross-reactivities between 84 and 134%. Without hydrolysis, lorazepam glucuronide gave less than 4% cross-reactivity in the assay.

Automated On-Line Hydrolysis of Benzodiazepines Improves Sensitivity of Urine Screening by a Homogeneous Enzyme Immunoassay

Benzodiazepines are used therapeutically as antidepressant, anxiolytic, anticonvulsant, hypnotic, muscle relaxant, and preanesthetic agents. Moreover, benzodiazepines are often abused for their euphoric effects. Consequently, benzodiazepines are commonly detected in toxicological analyses. Immunoassays of benzodiazepines are plagued by several factors that make reliable detection difficult. Complicated metabolic pathways, including N -dealkylation, C -hydroxylation, and glucuronidation, effectively reduce the parent compound to more polar, and thus easily excreted, metabolites. Some of these metabolites are pharmacologically active; in some cases, they are present in higher concentrations and have a longer elimination half-life than the parent compound (1)(2). Many, especially the glucuronide conjugates, which appear at high concentrations in the urine, do not readily cross-react with commercial immunoassays. In contrast, other benzodiazepine metabolites demonstrate high cross-reactivity, depending on the immunoassay utilized (3). In the US, 13 benzodiazepines of various potencies are commonly prescribed. Therapeutic daily doses have substantially decreased with the newer generation of benzodiazepines (2). Variations in dosage and in ultimate excretion patterns complicate the ability to accurately detect benzodiazepine use (4). Accumulated research findings indicate pretreating urine specimens with β-glucuronidase (EC 3.2.1.31) increases the diagnostic …