Differential regulation of endogenous glucose-6-phosphatase and phosphoenolpyruvate carboxykinase gene expression by the forkhead transcription factor FKHR in H4IIE-hepatoma cells.

The insulin responsive H4IIEC3 rat hepatoma cell line (H4 cells) was used in order to determine the role of the transcription factor FKHR in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Both PEPCK and G6Pase contain putative FKHR binding sites in their promoter sequence. Using a retroviral expression system, we stably overexpressed FKHR in H4-cells. FKHR was phosphorylated in a PI 3-kinase- and Akt-dependent manner, and was translocated from the nucleus to the cytoplasm in response to insulin. Furthermore, overexpression of FKHR markedly increased the expression of the catalytic subunit of G6Pase (basal about 2.5-fold, dexamethasone/cAMP stimulated about fivefold, respectively). In contrast, both basal and dexamethasone/cAMP-induced levels of PEPCK mRNA were unaffected by FKHR-overexpression. These data suggest a specific function for FKHR in the regulation of hepatic gluconeogenesis at the level of G6Pase, but not PEPCK gene expression.

Analysis of underivatized amphetamines and related phenethylamines with high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Amphetamine, methamphetamine, illicit designer phenethylamines (MDA, MDEA, MDMA, MBDB, and BDMPEA), and other phenethylamines (benzyl-1-phenylethylamine, cathinone, ephedrine, fenfluramine, norfenfluramine, phentermine, 1-phenylethylamine, phenylpropanolamine, and propylhexedrine) were extracted from serum using a solid-phase extraction procedure. The extracts were examined with high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). The drugs were separated on ODS column in acetonitrile/50 mM ammonium formate buffer (pH 3.0) (25:75) as a mobile phase. Full-scan mass spectra of drugs examined by means of APCI with collision-induced dissociation showed protonated molecular ions and fragments typical for particular drugs. LC-APCI-MS allowed an unequivocal differentiation of all drugs involved. The quantitation was performed using selected ion monitoring of protonated molecular ions and fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 1 to 5 microg/L serum, and the recoveries ranged from 58 to 96%. A linear response was observed for all drugs in the range from 5 to 500 microg/L. The method was applied for routine determination of amphetamine, MDMA, MDA, and MDEA in one run. Solid-phase extraction used assured simultaneous isolation of various groups of basic drugs of forensic interest (opiates, cocaines, phenethylamines, and benzodiazepines) from biofluids.

Monitoring of olanzapine in serum by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A selective assay of olanzapine with liquid chromatography atmospheric pressure chemical ionization (LC-APCI-MS, positive ions) is described. The drug and internal standard (ethyl derivative of olanzapine) were isolated from serum using a solid-phase extraction procedure (C18 cartridges). The separation was performed on ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (25:75). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection (SIM) was applied with the following ions: m/z 313 and 256 for olanzapine and m/z 327 and 270 for the internal standard for quantitation. The limit of quantitation was 1 microg/l, the absolute recovery was above 80% at concentration level of 10 to 100 microg/l. The method tested linear in the range from 1 to 1000 microg/l and was applied for therapeutic monitoring of olanzapine in the serum of patients receiving (Zyprexa) and in one case of olanzapine overdose. Olanzapine in frozen serum samples and in frozen extracts was stable over at least four weeks. The examinations of urine extracts from patients receiving olanzapine revealed peaks of postulated metabolites (glucuronide and N-desmethylolanzapine).

Poor reproducibility of in-source collisional atmospheric pressure ionization mass spectra of toxicologically relevant drugs.

The purpose of the study was to examine the intra- and interlaboratory reproducibility of mass spectra obtained with liquid chromatography-atmospheric pressure ionization mass spectrometry (LC--API-MS) both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) modes. Toxicologically relevant drugs of different polarity were selected as test substances: morphine-6-glucuronide, 6-monoacetylmorphine, codeine, lysergic acid diethylamide, methylenedioxymethamphetamine. The study was performed in two laboratories using identical instruments and in one using a slightly different instrument. Basic instrument settings and mobile phase were identical in all laboratories. Mass spectra of drugs were taken at four collision energy voltages and using mobile phase of different composition (four concentration levels of acetonitrile and of ammonium formate buffer). The experiments demonstrated that mass spectra of given drugs, obtained in identical conditions with identical instruments, may show very different degrees of fragmentation. Mass spectra obtained with different instruments differed profoundly not only in the degree of fragmentation, but also different fragments and adducts were observed. Short-term intralaboratory reproducibility of mass spectra was satisfactory. On the other hand, the long-term experiments showed different degrees of fragmentation of APCI-generated mass spectra at nominally identical fragmentation energy. The changes in the composition of the mobile phase (concentration of organic modifier or buffer molarity) did not affect the reproducibility of fragmentation to any relevant degree. The study showed that the interlaboratory exchange and use of mass spectrum library, generated by single-quadrupole (LC--API-MS instruments, is hardly feasible at the moment, even under very carefully standardized conditions.

Determination of common drugs of abuse in body fluids using one isolation procedure and liquid chromatography--atmospheric-pressure chemical-ionization mass spectromery.

A method for determining opiate agonists (morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-monoacetylmorphine, codeine, codeine-6-glucuronide, dihydrocodeine, dihydromorphine, buprenorphine, methadone, tramadol, and ibogaine), cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester) and lysergic acid diethylamide in serum, blood, urine and other biological matrices is presented. Aliquots (0.5-1.5 mL) of biological fluids were spiked with appropriate deuterated internal standards and extracted using a common solid-phase extraction method (C18 cartridges). The extracts were subjected to liquid chromatographic-atmospheric-pressure chemical-ionization mass spectrometric examination using selected ion monitoring procedures. These procedures were developed after analysis of full-scan mass spectra of examined compounds. The extraction method appeared very universal; the recoveries were high for almost all drugs and the extracts were very clean. The procedure was applied for routine forensic casework.

Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d3 and 7-AF-d3 were used as internal standards. The drugs were separated on ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d3 and F-d3), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d3), 330 (3-OHF). The limits of detection were: 0.2 microg/l for F and 7-AF, 1 microg/l for N-DF and 3-OHF. The method was linear in the range 1-500 microg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC-APCI-MS seems to be a method of choice for these compounds.