Development and clinical application of high performance liquid chromatography for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine.

A high performance liquid chromatography (HPLC) method has been developed for the simultaneous determination of plasma levels of theophylline and its metabolites without interference from caffeine or caffeine metabolites. The method is simple and of practical use because it is applicable even to plasma samples from patients who take caffeine-containing beverages. The method was also reproducible with a coefficient of variation of less than 5% for each analyte. The levels of theophylline, determined by HPLC, were validated by their high correlation to the levels obtained by fluorescence polarization immunoassay. HPLC was used to determine theophylline levels in patients with bronchial asthma. The data revealed that the ratio of 1,3-dimethyluric acid, the major metabolite of theophylline, to theophylline concentration in the plasma was within a narrow range in most patients (0.055 +/- 0.01, n = 66), regardless of the method of theophylline administration or the time of blood sampling. Conversely, this ratio was as low as 0.027 +/- 0.005 in the patient with a long plasma half-life of theophylline. These results suggest that it may be possible to predict the plasma half-life of theophylline for each patient from a single blood sample. This may be useful when planning theophylline administration, especially in patients with abnormal theophylline metabolism.

Development of an efficient amino acid sequencing method using fluorescent Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

In this paper, a new method is described for N-terminal amino acid sequencing of peptides using the fluorescent reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS). Sequence determination is carried out by identifying thiazolinone (TZ) amino acids, which are generally unstable and difficult to detect. The employed system can easily and quickly derive TZ amino acids using the Edman reaction with DBD-NCS; these amino acids are also stable enough to be efficiently detected by high-performance liquid chromatography. Resultant detection limits for DBD-TZ amino acids range from 50 fmol to a sub-picomole level (S/N = 3). This system successfully analyzed sequences of Leu5-enkephalin (25 pmol) and angiotensin I (100 pmol) using fluorometric detection at 524 nm with excitation at 387 nm.

Sensitive determination of enantiomers of amino acids derivatized with the fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole, separated on a Pirkle-type column, Sumichiral OA 2500(S).

Some L- and D-amino acids (Phe or Leu) were derivatized with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a Pirkle-type column, Sumichiral OA 2500(S) [(S)-1-naphthylglycyl-3,5-dinitrophenylamide silica gel] with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection was made at 530 nm with excitation at 470 nm. No racemization of the enantiomers occurred during the derivatization reaction. The separation factors (alpha) for NBD-L-Phe and NBD-D-Phe, and NBD-L-Leu and NBD-D-Leu, were 1.27 and 1.17, respectively. The detection limits were in the range of ca. 30 fmol.

Synthesis of novel fluorogenic Edman reagents, 7-N,N-dimethylaminosulphonyl- 4-(2,1,3-benzoxadiazolyl)isothiocyanate (DBD-NCS) and 7-aminosulphonyl-4-(2,1,3-benzoxadiazolyl)isothiocyanate (ABD-NCS).

Novel fluorogenic Edman reagents, 7-N,N-dimethylaminosulphonyl-4- (2,1,3-benzoxadiazolyl)isothiocyanate (DBD-NCS) and 7-aminosulphonyl-4-(2,1,3-benzoxadiazolyl)isothiocyanate (ABD-NCS) having no fluorescence themselves were synthesized. The derivatives of amino acids with DBD-NCS fluorescence at 505 nm with excitation at 385 nm. They were separated on a reversed-phase HPLC column and detected at the sub-picomol level. Ala-Phe derivatized with DBD-NCS was cleaved by acid to generate DBD-thiocarbamyl-Ala.

Determination of medroxyprogesterone acetate in serum by HPLC with peroxyoxalate chemiluminescence detection using a fluorogenic reagent, 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole.

The high-performance liquid chromatographic determination of medroxyprogesterone acetate (MPA) with peroxyoxalate chemiluminescence (PO-CL) detection is described. The spiked serum containing MPA was extracted on Bond-Elut C18 columns and derivatized with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). The hydrazone of MPA with DBD-H was confirmed to be a mono-DBD-derivative. The reaction mixture was separated by direct injection onto a C18 analytical column, and quantified by PO-CL detection. The linear range of the standard curve, in serum, was 15.6-96.6 ng ml-1 with a detection limit of 9 ng ml-1 using only 100 microliters of serum, while the detection limit of standard MPA derivatized with DBD-H was 8.7 fmol per injection. The relative standard deviation of the method was 7.4% at 19.3 ng and 1.7% at 77.3 ng ml-1.

High performance liquid chromatography with chemiluminescence detection of methamphetamine and its related compounds using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole.

A high performance liquid chromatographic assay of methamphetamine (MP) and its related compounds, i.e. ephedrine (EP), norephedrine (NE), p-hydroxymethamphetamine (p-HMP), p-hydroxyamphetamine (p-HAP) and amphetamine (AP), with peroxyoxalate chemiluminescence detection has been developed. 4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was used as a fluorescent labeling reagent. A mixture of hydrogen peroxide and bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate in acetonitrile was used as a postcolumn chemiluminogenic reagent. DBD derivatives of MP and its related compounds were separated by a gradient elution with acetonitrile and 0.01 M imidazole buffer (pH 7.0) within 65 min. The detection limits (S/N = 3) for the proposed method for MP, AP, EP, NE, p-HMP and p-HAP were 27, 100, 40, 133, 25 and 133 fmol on column, respectively. The recoveries of these compounds with normal urine samples were 87.4-106.4%. The method was successfully applied to the assay of MP and its metabolites in urine samples from MP addicts. A good linear correlation for the resulted amounts of MP or AP between the proposed method and gas chromatography was obtained (r = 0.993 for MP or 0.991 for AP).

Precolumn fluorescence tagging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7-aminoalkylamino-2,1,3-benzoxadiazoles.

Four new 2,1,3-benzoxadiazole amine reagents having different functional groups at the 4- and 7-positions, [4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-(N,N-dimethylaminosulphonyl)-7-N-piperazino-2,1,3-benzoxad iazole (DBD-PZ), 4-(N,N-dimethylaminosulphonyl)-7-N-cadaverino-2,1,3-benzoxad iazole (DBD-CD) and ammonium 7-N-piperazino-2,1,3-benzoxadiazole-4-sulphonate (SBD-PZ)] were synthesized as fluorogenic tagging reagents for carboxylic acids in high-performance liquid chromatography. The reagents, except SBD-PZ, reacted with carboxylic acid at room temperature in the presence of activation agents to produce fluorescent adducts. The maximum wavelengths of arachidic acid tagged with DBD-PZ, DBD-CD and NBD-PZ were 569 nm (excitation, 440 nm), 561 nm (excitation, 437 nm) and 541 nm (excitation, 470 nm), respectively. Among various activation agents tested [diethyl phosphorocyanidate (DEPC), diphenyl phosphoroyl azide (DPPA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-pyridine, 2.2'-dipyridyl disulphide-triphenylphosphine (Mukaiyama A) and 2-chloro-1-methylpyridinium iodide-triethylamine (Mukaiyama B)], DEPC and Mukaiyama A were more effective than the others. When the piperazino reagents (DBD-PZ and NBD-PZ) were used as the tagging reagents, the derivatization reaction in the presence of Mukaiyama A was faster than that in the presence of DEPC. Although the reaction in the presence of Mukaiyama A was completed after 30 min, an unknown peak derived from the activation agent appeared on the chromatograms. The fluorescence peak intensities were compared in the presence of DEPC. The order of the fluorescence peak areas obtained after reaction for 6 h in the presence of DEPC was DBD-PZ greater than DBD-CD greater than NBD-PZ. Thirteen saturated free fatty acids (FFAs) derivatized with DBD-PZ (or DBD-CD) and DEPC (or Mukaiyama A) in acetonitrile were separated completely by linear gradient elution on a reversed-phase ODS column. Eight drugs (ibuprofen, indomethacin, dinoprost, prostaglandin E1, dehydrocholic acid, ursodesoxycholic acid, hydrocartisone succinate and prednisolone succinate) were also tagged with DBD-PZ in the presence of DEPC and separated by isocratic elution. The detection limits (signal-to-noise ratio = 3) of FFAs tagged with DBD-PZ were in the range 3.2-4.7 fmol, whereas those of drugs were in the range 3.9-14 fmol.

Use of 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a labelling reagent for peroxyoxalate chemiluminescence detection and its application to the determination of the beta-blocker metoprolol in serum by high-performance liquid chromatography.

Fluorogenic reagents having a benzofurazan moiety, viz., 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 7-fluoro-4-nitro-2,1,3-benzoxadiazole and 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole, were compared for the sensitive analysis of their derivatives by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection. Of the proline derivatives, DBD-proline was the most sensitive with a detection limit of 2 fmol. The optimum concentrations of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and H2O2 for the post-column reaction were 0.5 and 75 mmol dm-3 respectively and amino acids and beta-blockers derivatized with DBD-F were detected in the range 0.2-40 fmol (signal-to-noise ratio = 3) using the proposed method. The lower detection limit of metoprolol (a beta-blocker having an isopropylamino group) spiked in serum was 0.8 ng ml-1 using 20 microl of serum (signal-to-noise ratio = 5).

4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole as chemilumigenic reagent for high performance liquid chromatographic peroxyoxalate chemiluminescence detection.

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), presented as a fluorogenic labelling reagent for amines and amino acids, is preferred for peroxyoxalate chemiluminescence (PO-CL) detection in high performance liquid chromatography. Amino acids and epinephrine derivatized with DBD-F were separated on a reversed phase column and detected at the femtomole level by the PO-CL detection system.

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate.

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole.

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.

Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins).

Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.

Wasp venom peptides; wasp kinins, new cytotrophic peptide families and their physico-chemical properties.

In addition to wasp kinins, the wasp venom contains a series of hydrophobic peptides, mastoparans and chemotactic peptides as major peptidergic components. The first major component in the venom is mastoparam. The peptides in the mastoparan family are tetradecapeptide amides which cause degranulation of the mast cells to release histamine from the cells, and act on the adrenal chromaffin cells to release catecholamines and adenylic acids. Some mastoparans cause hemolysis and serotonin release from the platelets. The new cytotrophic peptides as the second major components are tridecapeptide amides possessing chemotactic activity for polymorphonuclear leucocytes and monocytes. Some of the peptides in this family also cause histamine release from the mast cells. Mastoparan takes a random coil structure in aqueous solution but changes its conformation to alpha-helix in methanolic solution or in the presence of lysophosphatidyl choline. This fact is confirmed also by the transferred nuclear overhauser effect by NMR analysis. The similar phenomenon was observed in the family of chemotactic peptides. The helical conformation of these peptides are amphipathic structure in which all of side chains of the hydrophobic amino acids are located on one side of the axis, and those of the basic or the hydrophilic amino acid residues are on an opposite side. Mastoparan enhances the membrane conductivity of the lipid bilayer when the peptide is investigated by the black lipid membrane experiment. This indicates that the peptide may be assembled in the membrane by changing its conformation and, for some reason, enhances the ion transfer through the membrane. These properties of the peptide may reveal various activities on the cell membrane.

[Urinary total estrogens in normal menstrual cycle and early pregnancy (author's transl)].

A ratio of urinary total estrogens to creatinine (E/C, micrograms/g) was measured in every morning through the cycle in normal menstruating cycling women and during her early pregnancy. The level of follicular phase was 17.7 +/- 5.4 micrograms/g, ovulatory phase 78.8 +/- 8.8 micrograms/g and luteal phase 26.7 +/- 11.2 micrograms/g. In the spontaneous pregnancies E/C levels elevated sharply after 15 weeks of gestation. In the pregnancy after induced ovulation with clomifene citrate and conjugated estrogens, E/C levels were decreased from 7 days prior to the initiation of genital bleeding of spontaneous abortion. The need for multiple determinations over considerable periods of time is solved by our methods of easily repeated sequential measurements of urinary total estrogens estimated by E/C ratio.