Contrasting effects of acute vs. chronic tricyclic antidepressant treatment on central morphine analgesia

Antinociception following central opioid microinjection in rats was assessed weekly via a tail-flick procedure during chronic tricyclic antidepressant (TCA) treatment. (1) Daily TCA: Subcutaneous injections of desipramine (DMI), 30 mg/kg, chlorimipramine (CMI), 10 mg/kg, or saline, 1 ml/kg, were given daily for 22 days. Morphine sulfate (M), 5 μg, was microinjected into the ventrolateral periaqueductal gray (VLPAG) at 7 day intervals. On day 1, DMI or CMI enhanced M analgesia whereas saline did not. Augmentation of M disappeared by days 8 and 15 for CMI and DMI, respectively and was replaced by attenuation which was still observed on day 22 for both TCAs. l-Tryptophan (LT), 100 mg/kg i.p., on days 15 and 22 temporarily restored TCA enhancement of M. Fourteen days after cessation of all daily TCA treatments, enhancement of M by CMI was similar to that observed on day 1, whereas recovery of DMI-induced facilitation was incomplete. (2) Weekly TCA: Weekly treatment with DMI, CMI, or saline in the same doses as above had similar effects. M analgesia was enhanced by the TCAs but not saline on day 1 ; this facilitation was absent by day 15. Attenuation of M by DMI or CMI was evident on day 22; 2 weeks after cessation of all weekly TCA treatments, complete recovery of TCA-induced augmentation was observed. Loss of M facilitation during chronic daily or weekly TCA administration may be related to reduction of central opioid and/or 5-HT₂ receptors.     

Use of Sep-pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis

A method is described for the rapid and quantitative extraction of free and conjugated steroids from urine using Sep-pak C18 cartridges. The method was evaluated by determining the efficiency of recovery of (1) radiolabeled steroid glucuronides, (2) radiolabeled steroids freed by enzymatic hydrolysis, (3) steroid sulphates, (4) selected reference neutral free steroids of varied structure and polarity, and (5) oestrogens. In all cases the cartridges gave results equal to or better than those obtained by solvent or Amberlite XAD-2 extraction methods. Each urine extraction could be completed in 2-3 minutes and no further purification of extracts was required prior to derivatisation and gas chromatographic analysis.     

High-speed liquid chromatographic determination of antiepileptic drugs in human plasma.

A high-speed liquid chromatographic method for the simultaneous determinations of diphenylhydantoin, phenobarbital and carbamazepine in human blood plasma is presented. This method involves two step extraction procedures with chloroform and uses 2 X 50 cm long stainless steel columns packed with a anion exchange resin, with a mobile phase of 4 mM ammonium phosphate buffer solution, pH 6.2 at a flow rate of 0.40 ml/min. The results presented show linear calibration curves and quantitative determinations as low as 1.0 mug of each drug added to 0.5 ml plasma. This method has a sensitivity sufficient to detect human plasma levels after therapeutic clinical doses.     

Thin-layer and gel permeation chromatographic separation of low molecular weight polyethylene glycol oligomers

We describe a gel permeation and a thin-layer chromatographic technique for the complete resolution of oligomers of ethylene glycol up to a chain length of fourteen ethylene oxide [OCH2CH2] units. We employed columns of Bio-Gel P-2 with 0.02 mol/l ammonium acetate as the eluant to prepare milligram quantities of each of the individual oligomers. Thin-layer chromatography on silica gel G plates with an ethyl acetate/methanol/water solvent provides a sensitive and simple method for monitoring multiple specimens. We determined the flame ionization detector response of purified individual ethylene glycol oligomers relative to tetraethylene glycol. These data permit the accurate quantification of the oligomeric profile of commercially available mixtures of low molecular weight polyethylene glycols (PEG). When PEG 400 is administered orally to normal subjects, aged three months through adulthood, they excrete in their urine a mixture of unmetabolized oligomers with a mean molecular weight of 360 +/- 14 daltons. The ability to measure absolute, rather than relative, amounts of ethylene glycol oligomers will permit more accurate studies of passive intestinal permeability in human subjects.     

Gas chromatographic determination of glibenclamide in plasma.

A gas-chromatographic method for glibenclamide determination in plasma is described. It involves derivatization of the drug with dinitrofluorobenzene and the use of an electron-capture detector. The quantitative evaluation is performed using tolbutamide as internal standard. Characteristics and specificity of the method for the principal metabolite of glibenclamide are examined.     

An improved extraction method for plasma steroid hormones.

An easy and rapid column chromatographic method for the extraction of steroid hormones from plasma is presented. It permits the nearly quantitative separation of the steroids in a single step with smaller expenditure of time and work as compared to the usual liquid-liquid extraction. Problems of emulsions are eliminated. Furthermore, fractionated separation of hormones from plasma is possible. A simple procedure for the selective extraction of estriol (isolated from estrone and estradiol) is described.     

High performance liquid chromatographic analysis of the antiarrhythmic drugs procainamide, disopyramide, quinidine, propranolol and metabolites from serum extracts

We describe a method for the simultaneous high performance liquid chromatographic determination of several antiarrhythmic drugs and some of their metabolites after extraction from 2.5 mL of spiked pooled sera. The extracts were applied to a C8 reversed phase column. Nine compounds of interest were resolved within the 30 minute run. An initial mobile phase of 80% phosphate (25 mmol/L, pH 3.5), 20% organic (acteonitrile:methanol, 2:3) was maintained for 2 min at which time a linear gradient was used to change the mobile phase to 30% phosphate, 70% organic at 20 min after injection. This composition was maintained from an additional 5 min. Absorbance at 212 nm was used for detection. Peak area ratios of drug to internal standard (N-propionylprocainamide) were used for quantitation. The relative standard deviations (and mean solute concentrations) of daily duplicate determinations for 15 days are: procainamide, 5.1% (5.9 mg/L); N-acetylprocainamide, 9.3% (6.0 mg/L); Mono-N-dealkyldisopyramide, 3.7% (4.1 mg/L); disopyramide, 4.3% (4.0 mg/L); quinidine, 4.5% (6.5 mg/L); and propranolol, 5.1% (0.097 mg/L). Dihydroquinidine and 4-hydroxypropranolol were also resolved but not quantitated.     

(2-Ethoxyethoxy)acetic acid: An unusual compound found in the gas chromatographic analysis of urinary organic acids

An unknown acidic compound was detected in a number of urine samples from patients with a suspected metabolic disorder and some patients treated with chemotherapy. The structure of this compound has been characterized as (2-ethoxyethoxy)acetic acid, using a gas chromatography/mass spectrometry/ computer system.The authentic compound was synthesized and compared with the unknown. Urinary (2-ethoxyethoxy)acetic acid is assumed to be formed endogenously from an exogenous precursor, probably 2-(2-ethoxyethoxy)ethanol.     

Determination of oxalates in plasma and urine using gas chromatography

A gas-chromatographic procedure for the analysis of oxalic acid is described. The procedure requires relatively small quantities of urine (1 ml) or plasma (5 ml). The procedure consists of three steps: (1) extraction of oxalic acid by acidified diethyl ether; (2) esterification by isopropanol; and (3) final analysis by gas chromatography.The oxalic acid was found to have an elution temperature of 107°C and a retention time of 14 min. The standard curve is linear up to 800 nmol. In the described condition the lower limit of detection is 20 nmol.The mean normal plasma and urine oxalate levels ($\text{x}\text{?}\text{± 2}\text{S.D.}$) were found to be 20 μmol/l ± 17.5 and 275 μmol/24 h ± 200 respectively.     

Special problems in the radioimmunoassay of plasma aldosterone without prior extraction and purification

A new method for the radioimmunoassay of plasma aldosterone is described. The assay is performed directly on plasma without extracting or purifying the aldosterone. The method fulfilled the usual requirements for the demonstration of accuracy. Extensive data on the properties of the antisera are given.     

Dynorphin: important mediator for electroacupuncture analgesia in the spinal cord of the rabbit

Intrathecal injection of 12 nmol of dynorphin elicited marked analgesia as measured by tail flick latency, the effect being about 20 times more potent than with morphine. This analgesic effect could be reversed by naloxone at a dose 1.5-fold higher than that needed to reverse morphine analgesia. Intrathecal injection of anti-dynorphin antibody blocked electroacupuncture (EA) analgesia by 77%, the effect lasting for at least 4 h. In rabbits made tolerant to EA analgesia by long-term EA stimulation, intrathecal injection of dynorphin no longer exhibited an analgesic effect. No analgesia was noticed when dynorphin (10 nmol) was injected into the periaqueductal grey (PAG) of the rabbit, nor was EA analgesia blocked by anti-dynorphin antibody injected into PAG. These results suggest that dynorphin reduces nocifensive responses in the spinal cord and may play an important role in mediating EA analgesia at the spinal level.     

The rapid extraction of paraquat from plasma using an ion-pairing technique.

A method for the quantitative extraction of paraquat from plasma is described. The method relies on the use of an organic-soluble ion pair of paraquat with dodecyl sulphate. The use of the extraction procedure in conjunction with the dithionite colour reaction gives a method which is suitable for emergency estimations with a sensitivity limit of 50 micrograms/l. The extraction procedure alone offers a new first step in the clean up of paraquat for analysis by other methods.     

Specific quantitation of plasma medroxyprogesterone acetate by gas chromatography/mass spectrometry

Investigation of various derivatives (O-methyloxime, trifluoroenol acetate and t-butyldimethylsilyl enol ether) coupled with the efficient preparation of a trideuterated analogue of medroxyprogesterone acetate, has allowed the development of a rapid and accurate assay for its quantitation in plasma by isotope dilution gas chromatography/mass spectrometry. High oral doses (2.8 g weekly) of medroxyprogesterone acetate are shown to lead consistently to plasma levels of less than 16 ng.ml-1.     

A sensitive and specific sandwich enzyme immunoassay for human thyroid-stimulating hormone

A sensitive and specific sandwich enzyme immunoassay (EIA) for human thyroid-stimulating hormone (hTSH) has been developed. hTSH is incubated with anti-hTSH IgG-coated polystyrene balls, and after washing they are further incubated with anti-hTSH Fab'-β-d-galactosidase conjugate. The β-d-galactosidase activity bound to the polystyrene balls is proportional to the amount of hTSH to be assayed. Polystyrene balls are coated with rabbit anti-hTSH IgG which had been affinity-purified and treated with human chorionic gonadotropin-Sepharose 4B to remove antibodies cross-reacting with structurally related hormones. Rabbit anti-hTSH Fab', which had been affinity-purified was conjugated with β-d-galactosidase from Escherichia coli using N,N′-o-phenylenedimaleimide.In the specific sandwich enzyme immunoassay developed, 1 nU (1 × 10^?9 U) of hTSH per tube can be measured and the sensitivity for serum hTSH level is 0.1 μU/ml when 10 μl of serum is used. No significant interference was observed in the presence of 1.3 mU hLH/tube, 0.5 mU hFSH/tube or 0.5 U hCG/tube. Recoveries of hTSH added to human sera were 95.3–104% with a standard deviation of 12.0–14.9%. The coefficients of within-assay and between-assay variations were 6.0–7.5% and 4.9–8.7%, respectively. The regression equation and coefficient for correlation to radioimmunoassay (RIA) were y (RIA) = 0.95x (EIA) + 3.2 and 0.97, respectively.Serum levels of hTSH in normal male and female adults were 2.4 ± 1.0 (SD) (n = 41) and 2.9 ± 1.3 (n = 46) μU/ml, respectively; those in hyperthyroidism and hypothyroidism were 0.28 ± 0.06 (n = 20) and 49.6 ± 24.7 (n = 22) μU/ml, respectively; and those in pregnant and postmenopausal women were 2.5 ± 1.2 (n = 7) and 2.7 ± 1.0 (n = 35) μU/ml, respectively, indicating that high serum levels of hCG or hLH and hFSH under these conditions did not significantly interfere with the present assay of hTSH at normal levels.