A simple and robust HPLC method for the determination of paroxetine in human plasma

The objective of this investigation was to establish and validate an HPLC method with UV detection for the determination of paroxetine in human plasma. The method was validated in the concentration range from 6 to 100 ng ml-1. The lower limit of reliable quantification (LLQ) was 6 ng ml-1. The extraction efficiency varied from 67.1 to 85.5% over this range. Accuracies calculated at three concentrations in each of three separate runs were between 99.4 and 109.6%, and precision data were between 1.86 and 9.1%. The means of the between-day precision at all concentrations were between 2.77 and 7.32%. The corresponding means of the accuracy data were in the region of 102.4 to 106.3%.     

Rapid gas chromatographic method for simultaneous determination of cholesterol and alpha-tocopherol in eggs

This paper focuses on the evaluation of a pilot community stroke service. Its aim is to illustrate how the insights gained from the research of two medical anthropologists, Kaufman and Pinder, provided an understanding of the experience of chronic illness in the community. Two linked themes identified in the literature, which also emerged from the exploratory phase of the evaluation, are discussed: the tension between the patient's goal of recovery and the professional's goal of rehabilitation; and the management of uncertainty. It is argued that it may be very difficult, if not impossible, to reconcile these divergent goals of recovery and rehabilitation, and that this problem is exacerbated for patients and professionals alike by the challenge of how to manage the uncertainty of a chronic illness such as stroke. The empirical findings of the first phase evaluation corroborate Kaufman and Pinder's findings. Furthermore, the present research suggests that the mismatch between the goals of recovery and rehabilitation, and the management of uncertainty are integrally related. The implications for practice are that there is a need for professionals to find ways of recognizing and taking account of the possible tensions between their goals and those of patients and families, and related to this, for the development of strategies which enable professionals to explain clearly the nature of stroke to patients and their carers.     

Delaying colostrum intake by one day impairs plasma lipid, essential fatty acid, carotene, retinol and alpha-tocopherol status in neonatal calves

To study whether a delayed start of colostrum feeding in calves affects plasma lipids, fatty acids and fat-soluble vitamins, one group was fed colostrum (milkings 1-4) on d 1 and 2, then mature milk up to d 7, whereas two other groups were fed glucose or water on d 1, colostrum (milkings 1-4) on d 2 and 3 and then mature milk up to d 7. In calves fed colostrum on d 1, starting 5-7 h after birth, plasma concentrations of triglycerides, phospholipids, total cholesterol and of essential and nonessential fatty acids in triglyceride, phospholipid and cholesterol ester fractions as well as of carotene, retinol and alpha-tocopherol up to d 7 were significantly higher than in calves in which colostrum feeding started after >24 h of life. On the other hand, plasma concentrations of vitamin B-6, vitamin B-12 and folic acid were not influenced. Results indicated reduced efficiency of absorption of colostral fatty acids and of fat-soluble vitamins, but not of (selected) water-soluble vitamins, if colostrum is not fed on d 1 of life. In conclusion, colostrum intake within the first 24 h of life is required for an adequate plasma lipid, essential fatty acid, carotene, retinol and alpha-tocopherol status in the first week of life of calves.     

A simple method for measurement of angiotensin II in plasma

A simple radioimmunological (RIA) method for the determination of angiotensin II in 0.5-1.0 ml samples of plasma is described and carefully evaluated. Before RIA was performed, the interfering plasma proteins were eliminated by ion exchange chromatography, and recovery from every column was checked with a small amount of [125I]angiotensin II. The sensitivity of the method was 4.0 ng/l; the coefficient of intra-assay variation was 10.0% and that of inter-assay variation 12.1%. Accuracy was studied both by adding various amounts of angiotensin II to plasma samples and by diluting plasma containing angiotensin II with the RIA buffer. Both studies gave very good correlations between found and expected values (r=0.998 and r=0.987). In a normal material (n=36), the mean angiotensin II concentration at 8 a.m. after 2 h ambulation 42.4 +/- 12.8 (S.D.) ng/l. Because the present method is accurate, precise, and practical, and allows measurement of angiotensin II in small samples, it seems useful for routine as well as for research work.     

Determination of morphine and codeine in plasma by HPLC following solid phase extraction

A cheap simple and rapid extraction procedure followed by a UV high performance liquid chromatography (HPLC) assay is described for the simultaneous determination of morphine (M) and codeine (C) in plasma. The method is based on extraction of these opiates from plasma using reversed phase (solid phase) extractions columns followed by HPLC with UV detection at 240 nm. The extraction step provides, respectively, 85 and 80% recovery for M and C. The response of the detection system is linear for both molecules in the studied range from 50 to 750 ng ml-1. No other drugs have been found to interfere with the assay. This method offers a quick, cost effective and reliable procedure for specifically determining M and C, from a small sample volume.     

A sensitive method for the determination of amitriptyline and nortriptyline in human plasma

The kinetics of the male gametogenesis during the pregonadal period, prespermatogenesis, and "early" spermatogenesis has been described in detail. Concerning spermatogenesis in the adult individual reference is made to the articles of Courot, Hochereau-de Reviers and Ortavant (1970) and of Clermont (1972). The comparison of female and male gametogenesis (Fig. 1) shows that the "gonia stage" (asterisks) of the female germ cells is limited to one proliferation wave only, whereas the "gonia stage" of the male germ cells consists of a first proliferation wave, comparable to that of oogonia, a preparative phase to initiate spermatogenesis, and a second proliferation wave with renewal and differentiation of the spermatogonia. Germ cells in the "gonia stage" are highly sensitive towards ionising radiation and cytostatic drugs.     

The simultaneous assay of progesterone, 17-hydroxyprogesterone, and deoxycorticosterone in human plasma by competitive protein binding

Testosterone oenanthate was administered intramuscularly in six infertile men with oligozoospermia and its effects on serum gonadotropins and some constituents in the seminal plasma were studied. One week after injection the mean serum FSH level was decreased to about 50%. Serum LH levels did not change. The mean ornithine decarboxylase activity in human semen was increased by 100% after the testosterone administration. The androgen dependent nature of ODC, fructose and sialic acid have been demonstrated.     

Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and alpha-tocopherol in liposomes

BACKGROUND: Familial juvenile polyposis (JP) is an autosomal dominant condition in which affected individuals develop upper or lower gastrointestinal (GI) juvenile polyps, or both, and have a predisposition to cancer of the gastrointestinal tract. The risk of GI cancer has not been well defined because of the small number of these families and the lack of follow-up. The objective of this study was to determine the prevalence and age at diagnosis of GI polyposis and cancer in a large JP kindred. METHODS: Medical records were reviewed, patients were interviewed, and histories were taken. Pathology reports and slides were reviewed by our pathologists. A database was created for analysis of clinical and pathologic factors. RESULTS: This kindred contains 117 members, 29 of whom have had upper or lower GI polyps or cancer, or both. All those affected have had colonic juvenile polyps or cancer, except for two who died of advanced gastric cancer and never had colonic evaluation. Nine individuals have had both upper and lower GI polyps or cancer. Sixteen of 29 (55%) affected patients have developed gastrointestinal cancer. Eleven (38%) have had colon cancer, and six (21%) have had upper GI cancers. CONCLUSIONS: The risk of gastrointestinal malignancy in affected members of this JP kindred exceeds 50%. The high risk of GI cancer warrants frequent endoscopic screening of both affected and at-risk family members. Screening will soon be facilitated by presymptomatic genetic testing for the identification of gene carriers.     

Analytical method development for the simultaneous quantitation of dexmedetomidine and three potential metabolites in plasma

Dexmedetomidine, a novel alpha 2-adrenergic receptor agonist, is being developed as an anesthetic adjunct for perioperative use. An assay method has been developed for the sensitive quantitation of dexmedetomidine and three metabolites in plasma: MPV-1305, MPV-1306 and MPV-1709. The method involves solid-phase extraction (C18 cartridge) of dexmedetomidine and metabolites followed by a two-step derivatization. The first step utilized BF3-MeOH to simultaneously mask a primary alcohol in MPV-1305 and a carboxylic acid in MPV-1306. The second step applied PFB-Cl to derivatize the imidazole ring for sensitive detection of these compounds by GC-negative chemical ionization MS at pg/ml concentrations. MPV-1709 was not derivatized in the process and was detected by GC-positive chemical ionization MS. Optimization of extraction and derivatization is discussed. The method is suitable for quantitation of dexmedetomidine, MPV-1305, MPV-1306 and MPV-1709 over concentration ranges of 0.1, 40, 0.5-100, 0.5-100 and 1.0-500 ng/ml, respectively. The method showed excellent specificity, linearity and sensitivity and is useful for profiling the pharmacokinetic disposition of these compounds.     

A tandem solid phase extraction, reversed-phase HPLC method for determining SDZ WAG 994 in dog, monkey and rat blood

The development and validation of a sensitive and specific HPLC method for SDZ WAG 994 (I) in dog, monkey and rat blood is described. Sample preparation entailed double solid phase extraction (SPE) of I and the internal standard from 0.5 ml of animal blood using a phenyl and propyl sulfonic acid cation exchange column, sequentially. Chromatographic separation was achieved on a YMC Basic C-8 narrowbore HPLC column and the eluates were detected by UV absorption at 266 nm. The method has a linear response up to at least 1800 ng/ml with a limit of quantification of 1 ng/ml across all species. Analysis of 'blinded' quality control dog and monkey blood samples over 3 or 4 days produced median precisions of 2.89 and 4.77%, and median reproducibilities of 4.86 and 10.9%, respectively. Curve fitting of variability estimates indicated that concentration independent error contributed 3-9% of the total method error for the tandem SPE procedure. Extracted endogenous material from blood matrices, several potential metabolites and cyclohexyladenosine were well resolved from the peaks of interest. The stability of I in dog blood stored at -20 degrees C is at least 6 months. The overall absolute and relative recovery of I using the tandem SPE procedure was 85.5 +/- 5.1% and 96.5 +/- 5.0%, respectively. The ruggedness of the method has been demonstrated by multiple analyses, from several toxicokinetic studies, performed by different analysts using comparable instrumentation.     

Solid-phase extraction with end-capped C2 columns for the routine measurement of racemic citalopram and metabolites in plasma by high-performance liquid chromatography

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C2 column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.     

Monitoring of tricyclic antidepressants in human serum and plasma by HPLC: characterization of a simple, laboratory developed method via external quality assessment

A reversed-phase high performance liquid chromatography (HPLC) method for the determination of plasma and serum levels of amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI) is described. The assay is based upon single step liquid/liquid extraction of these compounds using hexane at pH 11 (recovery between 92 and 105%), a Nova-Pack C-18 HPLC cartridge column, a mobile phase composed of a phosphate buffer with 50% (v/v) acetonitrile and about 0.2% (v/v) diethylamine (final pH: 8) and solute detection at 242 nm. Using 1 ml of plasma or serum and econazole as internal standard, drug levels between 20 and 400 ng ml(-1) (about 60-1450 nM) were found to provide linear calibration graphs. For drug concentrations in the range of 70-120 ng ml(-1) (about 240-430 nM), intraday and interday imprecisions (n = 5) were determined to be < 6.0, and < 15%, respectively. Data reported include those gathered over a 3-year period during which this assay was employed for therapeutic drug monitoring and clinical toxicology. The performance of the laboratory developed assay was assessed via analysis of monthly samples provided by an external quality control scheme.     

3-Hydroxyanthranilic acid is an efficient, cell-derived co-antioxidant for alpha-tocopherol, inhibiting human low density lipoprotein and plasma lipid peroxidation

alpha-Tocopherol (alpha-TOH) can promote lipid peroxidation in human low density lipoprotein (LDL) unless co-antioxidants are present that eliminate the chain-carrying alpha-tocopheroxyl radical (alpha-TO.) (Bowry, V. W., Mohr, D., Cleary, J., and Stocker, R. (1995) J. Biol. Chem. 270, 5756-5763). Interferon-gamma inhibits human monocyte/macrophage-facilitated LDL lipid peroxidation via induction of cellular tryptophan degradation and production and release of 3-hydroxyanthranilic acid (3HAA) (Christen, S., Thomas, S. R., Garner, B., and Stocker, R. (1994) J. Clin. Invest. 93, 2149-2158). We now report on the mechanism of antioxidant action of 3HAA. 3HAA directly reduced alpha-TO. in UV-exposed micellar dispersions of alpha-TOH or in LDL incubated with soybean 15-lipoxygenase (SLO), as assessed by electron paramagnetic resonance spectroscopy. 3HAA did not inhibit SLO enzyme activity. Anthranilic acid, which lacks the phenoxyl group, was incapable of reducing alpha-TO.. 3HAA dose-dependently inhibited the peroxidation of surface phospholipids and core cholesteryl esters in LDL exposed to SLO, peroxyl radicals (ROO.), or Cu2+; oxidants that convert alpha-TOH to alpha-TO.. In all cases, sparing of LDL's alpha-TOH, but not ubiquinol-10 (CoQ10H2), was observed until the majority of 3HAA was consumed. Addition of 3HAA or ascorbate prevented further consumption of alpha-TOH and accumulation of lipid hydroperoxides when added to aqueous or lipophilic ROO.-oxidizing LDL after complete and partial consumption of CoQ10H2 and alpha-TOH, respectively. In contrast, addition of urate, an efficient ROO. scavenger incapable of scavenging alpha-TO., did not efficiently inhibit ongoing lipid peroxidation. Oxidation of 3HAA-supplemented human plasma by aqueous ROO. resulted in the successive consumption of ascorbate, CoQ10H2, 3HAA, bilirubin, alpha-TOH, and urate. Lipid peroxidation was prevented as long as ascorbate, CoQ10H2, and 3HAA were present, but subsequently proceeded as a free-radical chain reaction concomitant with alpha-TOH, bilirubin, and urate consumption. Addition of 3HAA to aqueous ROO.-oxidizing plasma, after complete consumption of ascorbate and CoQ10H2, strongly inhibited ongoing lipid peroxidation and consumption of alpha-TOH, bilirubin, and urate immediately and as efficiently as did ascorbate. These findings demonstrate that 3HAA is a highly efficient co-antioxidant for plasma lipid peroxidation by virtue of its ability to interact with alpha-TO. in lipoproteins. Since interferon-gamma is the principal inducer of tryptophan degradation and release of 3HAA by monocytes/macrophages, this may represent a localized extracellular antioxidant defense against LDL oxidation in inflammation.     

An automated method for the simultaneous determination of pravastatin and its main metabolite in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

An unexpected t(1;19) translocation is described in a fetus. Inherited from the mother, this translocation was found during the course of a normal prenatal diagnosis made for maternal age. The very short length of chromosomal translocated segments and their labelling pattern made high-resolution cytogenetic methods and fluorescence in situ hybridization techniques necessary for the correct identification of this karyotype rearrangement, both in mother and fetus. Different modes of meiotic segregation, leading to potential erroneous prenatal diagnoses, are discussed.     

New method for simultaneous measurement of viscosity,density and surface tension of metallic melts at high temperatures

A new method for determination of the viscosity of metallic melts based on the measurement of the velocity of gas bubbles in the melt, is developed. Gas bubbles are produced at known depth through a capillary tube. The time needed by the bubble to reach the surface of the melt is measured with the help of a laser beam. The method is tested with reference metals (Sn, Pb, Cu). The results show that there is a large dependence of the bubble velocity on the viscosity although the diameter of the bubbles is large. The recorded pressure curve in the capillary tube during the bubble formation allows the measurement of the surface tension and the density of the metallic melt to be determined by using the method of the maximum bubble pressure.     

Determination of dehydroepiandrosterone sulfate in human plasma by gas chromatography/mass spectrometry using a deuterated internal standard: a method suitable for routine clinical use

A capillary gas chromatographic-mass spectrometric method for the determination of dehydroepiandrosterone sulfate (DHEA-S) in human plasma is described. [7,7-2H2]DHEA-S is used as internal standard. After desulfation by methanolysis, heptafluorobutyryl derivatives are prepared, and selected ion monitoring of characteristic fragment ions--m/z 270 for DHEA and m/z 272 for [7,7-2H2]DHEA--carried out. Requiring small amounts of plasma, the rapid, convenient work-up and the application of bench-top GC/MS instrumentation proved our method to be suited for routine clinical use in adults and children. The method needs no complex corrections for isotope contributions and provides good accuracy and precision. Comparative values of samples assayed by our GC/MS procedure and by a direct RIA indicated that the RIA overestimates the true concentration.     

A method for simultaneous determination of plasma and erythrocyte antioxidant status. Evaluation of the antioxidant activity of vitamin E in healthy volunteers

1. Since oxygen free radicals are directly involved in a variety of pathologies such as atherosclerosis, diabetes mellitus, inflammation and/or when a deficit of defences of the organism against radicals occurs, we developed a suitable and simple method to determine both the erythrocyte sensitivity to an oxidative stress and plasma antioxidant protective capacity. 2. This test is based on the introduction at 37 degrees C of a radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), within an erythrocyte suspension leading to a membrane alteration and ultimately to haemolysis. The latter can be quantified by determining the lacticodeshydrogenase activity released in the medium. The erythrocyte sensitivity to haemolysis and the volume of plasma inhibiting 50% of the haemolysis were determined. 3. Intra-assay CVs were 1.9% for erythrocyte sensitivity to oxidative stress and 3.4% for inhibitory 50% plasma volume. Inter-assay CVs for both erythrocyte sensitivity and inhibitory 50% plasma volume were 4%. 4. The reliability of this method was assessed and applied to test the protective effect of vitamin E, a well known antioxidant agent, in six healthy volunteers. Two weeks after daily administration of 500 mg of vitamin E, the mean plasma vitamin E concentration increased by 41% from 10.7 +/- 2.0 mg l-1 before treatment (P < 0.05). As the vitamin E concentration increased, the mean inhibitory 50% plasma volume and the percentage of haemolysed erythrocytes decreased respectively by 29% from 3.35 +/- 0.5 microliter (P < 0.05) and 18% from 71.5 +/- 3.8% (P < 0.05). No significative variation of these parameters was observed in six adult men without vitamin E supplementation. 5. Thus, this global and simple test permits an antioxidant status evaluation of a patient. It can be applied to various pathologies and allows the potency of new antioxidant molecules to be evaluated.