Dual temperature- and pH-responsive fluorescence molecular probe for cellular imaging utilizing a PNIPAAm-fluorescein copolymer.

The thermally controlling fluorescence intensity of a molecular probe for cellular imaging has been investigated. A reversible temperature-induced phase transition of N-isopropylacrylamide/fluorescein copolymers [poly(NIPAAm-co-FL)] was used as a molecular switch to control the fluorescence intensity of the imaging probe. The copolymer displayed environmentally sensitive fluorescence properties, in which the fluorescence intensity changed with the response to both the temperature and the pH. Utilizing these features, we monitored the thermal-aggregation process of BSA by the fluorescence resonance energy transfer (FRET) method. Additionally, the cellular uptake in RAW264.7 cells of poly(NIPAAm-co-FL) conjugated with lipid was studied using a confocal laser scanning microscope.

Aqueous chromatographic system for separation of biomolecules using thermoresponsive polymer modified stationary phase.

We have investigated a new method for HPLC using packing materials modified with a functional polymer, such as thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm). PNIPAAm-modified silica exhibits temperature-controlled hydrophilic-hydrophobic surface property changes in aqueous systems. Temperature-responsive chromatography is performed with an aqueous mobile phase without using an organic solvent. We designed ternary copolymers of NIPAAm introduced 2-(dimethyl-amino) ethyl methacrylate (DMAEMA) as a cationic monomer and butyl methacrylate (BMA) as a hydrophobic monomer. A cationic thermoresponsive hydrogel grafted surface would produce an alterable stationary phase with both thermally regulated hydrophobicity and charge density for separation of bioactive compounds. In this study, we achieved successful separation of lysozyme without the loss of bioactivity by temperature-responsive chromatography. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase, thus the separation system would have potential applications in the separation of biomolecules.

Triage test and HPLC assay for analysis of vegetamin, an antipsychotic agent, using gastric contents and blood specimens.

Vegetamin is an antipsychotic agent composed of phenobarbital, promethazine and chlorpromazine. While phenobarbital can be detected by the Triage kit for screening drugs of abuse, the other two components cannot. We describe here the detection of promethazine and chlorpromazine using either a high performance liquid chromatography (HPLC) unit equipped with an ultraviolet, a photodiode array (PDA), or an electrochemical detector (ECD), or gas chromatograph-mass spectrometry (GC-MS). The absorption and mass spectra of promethazine and chlorpromazine were nonspecific, and thus the ECD-HPLC system was best equipped to quantitate these components. Comparison of the chromatograms of blood and gastric contents aids the identification of drug metabolites.

Direct determination of glucuronide and sulfate of p-hydroxymethamphetamine in methamphetamine users' urine.

Two conjugates of p-hydroxymethamphetamine (p-OHMA), p-OHMA-glucuronide (p-OHMA-Glu) and p-OHMA-sulfate (p-OHMA-Sul) have been identified in methamphetamine (MA) users' urine by using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS-MS). The synthesis of p-OHMA-Glu and p-OHMA-Sul, and an LC-MS procedure for the simultaneous determination of MA and its four metabolites, amphetamine (AP), p-OHMA, p-OHMA-Glu and p-OHMA-Sul, in urine have also been established. After deproteinizing urine samples with methanol, LC-MS employing a C(18) semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Based on the established method, p-OHMA-Sul was detected at higher concentrations than p-OHMA-Glu in all of the three urine samples tested. These data suggest that sulfation is a major pathway in the MA phase II metabolism.

Metabolism of the psychotomimetic tryptamine derivative 5-methoxy-N,N-diisopropyltryptamine in humans: identification and quantification of its urinary metabolites.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.

Mechanistic studies on the decomposition of sodium cyanide in aqueous solution and in the solid state.

The mechanism of the spontaneous decomposition of sodium cyanide in aqueous solution and in the solid state was studied by ion chromatography (IC), FT-Raman spectroscopy, gas chromatography/mass spectrometry (GC/MS), and carbon-13 nuclear magnetic resonance spectroscopy (13C NMR). In the aqueous solution, gradual decomposition of the cyanide to carbonate by a displacement reaction was observed. In the solid state, sodium cyanide was found to be stable when kept in dry air, however, it decomposed by the same mechanism as that in aqueous solution under non-dry conditions.

Liquid chromatography-mass spectrometric and liquid chromatography-tandem mass spectrometric determination of hallucinogenic indoles psilocin and psilocybin in "magic mushroom" samples.

Accurate and sensitive analytical methods for psilocin (PC) and psilocybin (PB), tryptamine-type hallucinogens contained in "magic mushrooms," were investigated using liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The chromatographic separation on an ODS column and mass spectral information gave complete discrimination between PC and PB without derivatization. The mass spectrometric detection had a high sensitivity, and the tandem mass spectrometric detection provided more specificity and accuracy, as well as high sensitivity. The detection limits ranged from 1 to 25 pg by LC-MS in the selected ion monitoring mode, and the intra- and inter-day coefficients of variation were estimated to be 4.21-5.93% by LC-MS-MS in the selected reaction monitoring mode. By applying the present LC-MS-MS technique to four real samples, the contents of PC and PB were found to vary over a wide range (0.60-1.4 and 0.18-3.8 mg/g dry wt. for PC and PB, respectively) between samples.

Development of simultaneous gas chromatography-mass spectrometric and liquid chromatography-electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine.

To prove the intake of recently controlled designer drugs, N-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), a simple, sensitive and reliable method which allows us to simultaneously detect BZP, TFMPP and their major metabolite in human urine has been established by coupling gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). GC-MS accompanied by trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed after the enzymatic hydrolysis and the solid phase extraction with OASIS HLB, and BZP, TFMPP and their major metabolites, 4'-hydroxy-BZP (p-OH-BZP), 3'-hydroxy-BZP (m-OH-BZP) and 4'-hydroxy-TFMPP (p-OH-TFMPP), have found to be satisfactorily separated on a semi-micro SCX column with acetonitrile-40 mM ammonium acetate buffer (pH 4) (75:25, v/v) as the eluent. The detection limits produced by GC-MS were estimated to be from 50 ng/ml to 1 microg/ml in the scan mode, and from 200 to 500 ng/ml in the selected ion monitoring (SIM) mode. Upon applying the LC-ESI-MS technique, the linear calibration curves were obtained by using the SIM mode for all analytes in the concentration range from 10 ng/ml to 10 microg/ml. The detection limits ranged from 5 to 40 ng/ml in the scan mode, and from 0.2 to 1 ng/ml in the SIM mode. These results indicate the high reliability and sensitivity of the present procedure, and this procedure will be applicable for proof of intake of BZP and TFMPP in forensic toxicology.

In vitro confirmation of selegiline N-oxidation by flavin-containing monooxygenase in rat microsome using LC-ESI MS.

In order to investigate the conversion of selegiline (SG), a drug used in the treatment of Parkinson's disease, to selegiline N-oxide (SGO) as a major metabolic pathway for SG, rat liver microsomal incubations were carried out in vitro in the presence of NADPH. SG was transformed into SGO in vitro as described in our previous human in vivo experiment. In the kinetic studies, the Vmax/Km value of the N-oxidation at pH 8 was found to be approximately four times greater than that at pH 7.4. The N-oxidation was also found to be inhibited by methimazole, an inhibitor of the flavin-containing monooxigenase (FMO) rather than by SKF 525A, an inhibitor of cytochrome P450s, and stimulated approximately two times by n-octylamine, an stimulator of FMO. Moreover, the N-oxidation activity remained almost unchanged in the presence of NADPH even after heating at 50 degrees C for a few minutes. The present data demonstrate that the N-oxidation of SG to SGO is principally mediated by FMO.

Extraction of thiamylal in serum using hydrophilic acetonitrile with subzero-temperature and salting-out methods.

For high-performance liquid chromatography (HPLC) of thiamylal, one of the barbiturates, the drug in serum samples was extracted by two alternative liquid-liquid extraction techniques using hydrophilic acetonitrile as a solvent and subzero-temperature and salting-out methods. Acetonitrile was mixed with the sample, separated by cooling at -20 degrees C or addition of sodium chloride, and injected directly into the HPLC apparatus. In both the methods, thiamylal was extracted effectively in the acetonitrile phase and pH adjustment of the sample was not required. The salting-out extraction method is rapid and would be suitable for quantitation of drugs in many samples. To avoid coextraction of added salt, the subzero-temperature extraction method was applied to identification of thiamylal by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry.

Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples.

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.

Simultaneous determination of eleven benzodiazepine hypnotics and eleven relevant metabolites in urine by column-switching liquid chromatography-mass spectrometry.

An automated column-switching liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometric (LC-APCI-MS) method for the simultaneous determination of frequently encountered benzodiazepine hypnotics and their relevant metabolites in urine has been established. A column-switching online solid-phase extraction technique was employed to enhance sensitivity and eliminate tedious sample pretreatment. The combination of an N-vinylacetamide-containing hydrophilic polymer online extraction column and a C18 semi-micro LC column provided the successful separations and MS determinations of the 11 benzodiazepines and 11 relevant metabolites tested in this study. The detection limits ranged 2-10 ng/mL and 10-50 ng/mL in the SIM and full-scan modes, respectively, using 50 microL of urine. The calibration curves were linear up to 500 ng/mL for all these analytes in the SIM mode. The present method provided successful forensic applications for the proof of benzodiazepine administration.