Oxytocin causes a long-term decrease of blood pressure in female and male rats

The aim of the present study was to investigate the long-term effects of oxytocin (OXY) on blood pressure (BP) and heart rate (HR) in conscious female and male rats. For this purpose, subcutaneous (SC) (0.01, 0.1, and 1 mg/kg) or intracerebroventricular (ICV) (1 microgram/kg) injections of OXY were given during 5-day periods. BP and HR were measured daily. A significant decrease in BP, without affecting HR, compared to saline-treated controls was seen in response to 0.1 (males: p < 0.01, females: p < 0.001) and 1 mg/kg (p < 0.001) of OXY given SC. BP gradually returned to preexperimental levels within 10 days after the last injection in male rats but, in females, the significant lowering of BP remained unchanged during this period. Also OXY ICV (1 microgram/kg) decreased BP (p < 0.05 after one day, p < 0.001 after 5 days of injections). This effect was still present 8 days after the last injection (p < 0.05). These results indicate that OXY may induce a long-term lowering of BP.     

Validation of a model predicting enrollment status in a chemoprevention trial for breast cancer

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], alpha-(cystein-S-yl)isovalerylurea [CVU], and alpha-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10 mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10 mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90-97.79% from the spiked liver homogenate and 89.68-96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.     

High-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry as a new tool for the determination of the mycotoxin zearalenone in food and feed

A new method for the determination of the mycotoxin zearalenone (ZON) in food and feed, based on HPLC-MS with an atmospheric-pressure chemical ionization (APCI) interface after extraction from cereals and clean-up by either conventional solid-phase or immunoaffinity cartridge is presented. The APCI interface parameters are optimized to provide detection of ZON with maximum sensitivity after RP separation of ZON on a C18 column with acetonitrile-water (40:60, v/v) at 1 ml/min column flow without split. Using APCI-MS detection, the sensitivity of the method was improved by a factor of ca. 50 in comparison to HPLC with fluorescence detection, allowing determination of ZON down to 0.12 microgram/kg maize which is well below present threshold values. Due to the selectivity of MS detection, it also was possible to quantitatively determine ZON both in raw extracts without clean-up using a normal-size (100 mm) chromatographic column or using only a short (20 mm) chromatographic column, when a clean-up was done to minimize possible interferences.     

Multiresidue liquid chromatographic method for determining residues of mono- and dibasic penicillins in bovine muscle tissues

A liquid chromatographic method with UV detection at 325 nm was developed for simultaneous determination of amoxicillin, ampicillin, penicillin G, and cloxacillin residues in bovine muscle tissue as their mercaptide derivatives. The penicillins are extracted from bovine tissues with 0.1 M phosphate buffer (pH 8.5), cleaned up on a t-C18 Sep-Pak cartridge, and eluted with 2 mL acetonitrile. After the acetonitrile in the eluate is evaporated to dryness, the residue is dissolved in 200 microL (40 + 60, v/v) acetonitrile-phosphate buffer (pH 6.5) and derivatized with acetic anhydride and mercuric chloride in the presence of 1,2,4-triazole at 65 degrees C for 30 min. Gradient analysis on a Spherisorb 5 microns ODS(2) (octadecyl silane) analytical column using a binary mobile phase consisting of acetonitrile and 0.10 M phosphate buffer (pH 6.5) in the presence of 0.0157 M sodium thiosulfate at 1 mL/min permits determination of each intact penicillin in bovine muscle tissue at > or = 10 ppb with recoveries > or = 72%. This laboratory method provides detection sensitivities equivalent to those of rapid tests used for screening beta-lactam drug residues in bovine tissue samples for regulatory enforcement.     

Serum homocysteine and methylmalonic acid in patients with rheumatoid arthritis and cobalaminopenia

This paper describes the synthesis of 14C-labeled glycosphingolipids using the reverse hydrolysis reaction (condensation) of sphingolipid ceramide N-deacylase. It was found that 50-70% of 14C-fatty acids were incorporated into various lyso-glycosphingolipids when a mixture of lyso-glycosphingolipids and fatty acids was incubated at 37 degrees C with 1 mU of the enzyme for 20 h in 1 ml of 25 mM phosphate buffer, pH 6.0-7.0, containing 0-0.1% Triton X-100. The optimum concentration of lyso-glycosphingolipids was 100-400 microM depending on the species of lyso-form when [14C]stearic acid was used at the concentration of 100 microM. Free 14C-fatty acids and lyso-glycosphingolipids were separated from the synthesized 14C-glycosphingolipids by using a Sep-Pak Plus Silica and a Sep-Pak CM or a QMA cartridge, respectively. After treatment of 14C-glycosphingolipids with endoglycoceramidase or sphingolipid ceramide N-deacylase, digestion products were clearly separated from the parent glycosphingolipids on TLC and determined using an image analyzer with a sensitivity 100 times higher than that using non-radiolabeled substrates. Using this method, we found endoglycoceramidase activity in a seaflower, Condylactis sp., for the first time.     

Blunted cortisol response after administration of corticotropin releasing hormone in endotoxemic dogs

To evaluate the effects of a standard inflammatory challenge on the dynamics of the hypothalamic-pituitary-adrenal (HPA) axis, we studied the effects of low-dose endotoxin (1.0 microgram/kg) on plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations in a saline-controlled study in five awake dogs. Four hours after endotoxin or saline challenge human corticotrophin-releasing hormone (hCRH; 1.0 microgram/kg) was administered. Plasma ACTH and cortisol levels increased considerably in response to endotoxin, from 13 +/- 1 ng/l to 360 +/- 85 ng/l (p < 0.01) and from 60 +/- 20 nmol/l to 710 +/- 80 nmol/l (p < 0.01). Despite a considerable difference in ACTH and cortisol levels prior to CRH administration between both studies (p < 0.01), the absolute increase in ACTH levels induced by hCRH was not different (231 +/ 43 ng/l vs 238 +/- 45 ng/l, control vs endotoxin). Plasma cortisol levels increased significantly in the control study (from 40 +/- 10 nmol/l to 330 +/- 40 nmol/l, p < 0.01), whereas they did not change in the endotoxin study after hCRH administration (from 710 +/- 80 nmol/l to 730 +/- 70 nmol/l, ns). We conclude that the HPA-axis reacts initially to endotoxin in such a way that cortisol, but not ACTH, secretion is maximized. Therefore, a blunted cortisol response to CRH testing is part of the initial response to infection.     

Acetonitrile as a possible marker of current cigarette smoking

Volatile nitriles are present in cigarette smoke. We tested the hypothesis that the presence of any of four nitriles in the blood can serve as a marker of recent cigarette smoking. We determined the sensitivity and specificity of these nitriles as indicators of daily cigarette smoking in 24 smokers (Group A) and 18 non-smokers (Group B), as well as the correlation between intensity of daily smoking and the blood concentration of acetonitrile. A new head space GLC assay method was used. Of the four nitriles, only acetonitrile was present in the blood of any study subject. Acetonitrile was moderately sensitive (67%) and entirely specific (100%) for self-reported daily smoking. There was fair correlation between blood acetonitrile concentration and the average daily number of cigarettes smoked (r2 = 0.39; P = 0.001), and the mean blood acetonitrile concentration was significantly higher (P = 0.03) among subjects with higher (> 10 cigarettes per day) current cigarette exposure (148.3 +/- 18.0 micrograms/l) than among smokers with low or minimal (1-10 cigarettes per day) exposure (43.3 +/- 6.0 micrograms/l). Thus, acetonitrile in blood appears to be highly specific and a moderately sensitive marker of cigarette smoking with a dose-effect relationship. As such, acetonitrile shows promise as a marker of current cigarette exposure.     

Simultaneous determination of five macrolide antibiotics in meat by high-performance liquid chromatography

A simple and rapid method using high-performance liquid chromatography (HPLC) for the simultaneous determination of five macrolides (josamycin, kitasamycin, mirosamicin, spiramycin and tylosin) in meat has been developed. The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3, v/v), and the extracts were cleaned up on a Bond Elut SCX (500 mg) cartridge. The HPLC separation was performed on a Puresil 5C18 column (150 x 4.6 mm I.D.) with a gradient system of 0.025 M phosphate buffer (pH 2.5)-acetonitrile as the mobile phase at a flow-rate of 1.0 ml/min. The drugs were detected at 232 mn for josamycin, kitasamycin, mirosamicin and spiramycin, and 287 mn for tylosin. The calibration graphs were rectilinear from 2.5 to 100 ng for each drug. The recoveries at the level of 1.0 microgram/g were 70.8-90.4%, and detection limits were 0.05 microgram/g for each drug.     

Changes in the concentrations of trace elements in human milk during lactation

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine 18 trace elements (Ba, Be, Bi, Cd, Co, Cs, Cu, La, Li, Mn, Mo, Pb, Rb, Sb, Sn, Sr, Tl, and Zn) in 55 human milk samples from 46 healthy mothers collected during lactation periods extending to 293 days after birth. Se was quantified by hydride generation atomic absorption spectrometry (HG-AAS). To test the accuracy and the precision of the analytical procedure, milk powder reference materials (BCR 063 and BCR 150) were analyzed. The results obtained by ICP-MS and HG-AAS showed good agreement with the certified values. Whenever available, trace element concentrations determined in the human milk samples were compared to reliable literature data. The concentrations of Be (< 0.05 to 0.9 microgram/kg), Bi (< 0.09 to 2.0 micrograms/kg), Cs (1.7 to 7.7 micrograms/kg), La (< 0.05 to 3.7 micrograms/kg), Rb (440 to 1,620 micrograms/kg), and Tl (< 0.08 to 0.5 microgram/kg) are the first to be reported for human milk. The concentrations of the essential trace elements Cu (p < 0.005), Mn (p < 0.05), Mo (p < 0.0005), Se (p < 0.001), and Zn (p < 0.0005) significantly decreased and the concentrations of cobalt significantly increased (p < 0.005) in human milk during the course of lactation. All concentrations for the essential trace element tin in the human milk samples were below the method detection limit of 0.3 microgram/kg. Among the not essential and toxic elements-with the exception of Ba, Pb, and Tl-the trend toward lower concentrations with continuing lactation is much less pronounced than for the essential trace elements. With the exception of Se, the daily intakes of essential trace elements of fully breast-fed infants are considerably lower than dietary recommendations.     

Release of proteins and polysaccharides from the albumen gland of the freshwater snail Helisoma duryi: effect of cAMP and brain extracts

The albumen gland is a compound tubular exocrine gland found in the female reproductive tract of freshwater pulmonate snails such as Helisoma duryi. It secretes a perivitelline fluid, composed of protein and polysaccharide complexes, and coats each fertilized egg. A 288-kDa native glycoprotein, composed of several 66-kDa subunits, was identified in soluble extracts of albumen gland. Forskolin stimulates the release of secretory granules, containing both proteins and polysaccharides, from the cytoplasm of the glandular cells. An acid extract of the central nervous system or the adenosine-3', 5'-cyclic monophosphate (cAMP) analogue 8-bromo cAMP, stimulates protein secretion from the gland. Pretreatment of the albumen gland with cAMP antagonist (Rp isomer of cAMP) inhibits the stimulatory effect of a brain extract. Digestion of brain extract with proteolytic enzymes abolishes its activity, suggesting the factor from the brain is peptidergic. The neuroactive agents serotonin, Phe-Met-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide, small cardioactive peptide B, and caudodorsal cell hormone were also tested for potential secretion-promoting ability. Brain extracts were partially purified with a Sep-Pak C18 reverse-phase cartridge and indicate the peptide is relatively hydrophobic. These results suggest that a brain peptide promotes the secretion of perivitelline fluid, and this is mediated by the adenylate cyclase/cAMP signal transduction pathway.     

Effects of pentagastrin and heptagastrin on acid secretion in man (author's transl)

Gastrin analogues are known to stimulate acid secretion with higher potency the more amino acids prolong the peptid chain towards the N-terminal end. In order to compare the response of pentagastrin (Gastrodiagnost, Merck, Darmstadt) and Heptagastrin (HOE 293, Hoechst AG, Frankfurt/M.) in 12 healthy subjects the two peptides were given subcutaneously in doses of 1 microgram/kg, 3 microgram/kg and 6 microgram/kg body weight. Additionally in 8 subjects 1 microgram/kg/h and 2 microgram/kg/h of the gastrins were given intravenously. Gastric content was aspirated under basal and stimulated conditions in portions of 15 minutes, volume (ml) and acid concentration (meq/l) of each fraction was measured and acid secretion (meq/90'), BAO and PAO were calculated. As compared with pentagastrin the dose response curve was shifted to the left, when heptagastrin was given subcutaneously. The amount of acid secretion over a time period of 90 min. was about 17% higher after heptagastrin than after pentagastrin. There was no difference between the peak acid outputs (PAO). Stimulation of acid secretion was prolonged after heptagastrin as compared to pentagastrin. Both gastrins acted similarly on all parameters measured when administered intravenously. Side effects as nausea and dizziness were observed in two subjects of each group.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A method is described which permits the trace analysis of 10 chlorobenzenes in aqueous samples. Chlorobenzenes were extracted from water samples by solid-phase extraction with a C18 cartridge and analysis was carried out by gas chromatography-mass spectrometry in the selected-ion monitoring mode. The recovery and precision of the method were evaluated by extraction of spiked reagent-grade water at concentration levels of 0.1, 1.0 and 10.0 micrograms/l. This method was applied to the determination of chlorobenzenes in tap, ground and river water. By preparing 200 ml of environmental water samples, the detection limits of the compounds studied were in the range of 0.010-0.042 microgram/l.     

Confirmation and quantitation of selected sulfonylurea, imidazolinone, and sulfonamide herbicides in surface water using electrospray LC/MS

A multianalyte method has been developed for the confirmation and quantitation of 16 selected sulfonylurea, imidazolinone, and sulfonamide herbicides in surface water. Samples are acidified, the analytes are extracted using RP-102 extraction cartridges, and the extracts are cleaned-up using an anion-exchange cartridge (SAX) stacked on top of an alumina cartridge. The final extracts are evaporated to dryness, redissolved in 10:90 or 20:80 acetonitrile/water, and analyzed using electrospray LC/MS. Confirmation criteria were defined so that identification of the analytes could be made with a reasonable degree of scientific certainty. Confirmation required that (1) LC/MS retention times of the analytes be within 1% of the retention times of the standards, (2) the molecular ion and two characteristic fragment ions per analyte be present, and (3) ion abundance ratios of the fragment ions relative to the molecular ion be within 20% of the ion ratios obtained for the standards. Each of the analytes in all of the samples used to validate this method met these confirmation criteria. Quantitation of the analytes at 0.1 and 1.0 ppb was demonstrated, with average recoveries between 70% and 114% and relative standard deviations that were less than 13%. The limit of quantitation (LOQ) for this method is 0.1 ppb (ng/mL) for all analytes. Six water types were used for the validation of this method.     

Ceranapril and cerebral blood flow autoregulation

In order to investigate the levels of the polycyclic aromatic hydrocarbons, mainly benzo[a]pyrene because of its carcinogenicity, 55 samples of smoke flavour and smoked foods were analysed. The samples tested included 11 samples of liquid smoke flavour and 44 samples of smoked foods like bacon, loin, turkey, sausage, ox rib, etc. from different brands. A liquid chromatographic method was developed using a fluorescence detector. Benzo[a]pyrene was found in 73% of the liquid smoke flavour samples analysed. The levels varied from 0.1 to 336.6 micrograms/kg. Three liquid smoke flavour samples showed levels of benzo[a]pyrene above the maximum level recommended by FAO/WHO (10 micrograms/kg). From the total of 44 smoked food samples analysed, benzo(a)pyrene was detected in 23 samples (52%). The levels varied from 0.1 to 5.9 micrograms/kg. Anthracene and fluoranthene, non-carcinogenic polycyclic aromatic hydrocarbons, were found in almost all the samples analysed. Benzo[ghi]perylene, 3,4-benzofluoranthene and 1,2,3,4-dibenzopyrene were not found in any of the 55 samples analysed.     

Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells

A method was developed to determine in eggs 2 components [4,6-dimethyl-2-hydroxypyrimidine and 1,3-bis(4-nitrophenyl)urea] of the anticoccidial drug nicarbazin, used to treat poultry. Samples were extracted with acetonitrile, and the extracts were washed with hexane and evaporated to dryness before analysis by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization. By switching from positive to negative ion monitoring and using gradient elution, both components were measured within one run. The limit of quantitation of the assay was 10 ng/g for each component. The results of a preliminary feeding trial in which chickens were fed contamination levels of the drug are also reported.     

Analysis of aromatic sulfonates in water by solid-phase extraction and capillary electrophoresis

The separation of 14 different aromatic sulfonates of environmental concern by capillary (zone) electrophoresis (CZE) is presented. A new off-line solid-phase extraction (SPE) enrichment procedure, that is compatible with CE analysis, was developed, using the styrene-divinylbenzene adsorbent LiChrolut EN. The combined method of SPE and CE allows the determination of aromatic sulfonates in water samples in the low microgram/l range. Separations are performed with a simple sodium borate buffer at pH 9.3. Analytes are detected by UV absorbance and fluorescence emission with a Xe-lamp excitation source, and both principles are compared. The recoveries for most of the sulfonates are > 70% for the extraction from spiked tap and river water. The average method precision is < 20% for replicate analyses. Very hydrophilic sulfonates cannot be extracted by this method. The detection limit of the combined method of SPE enrichment and CE analysis is approximately 0.1 microgram/l for 200-ml water samples. The performance of the method was checked with the analysis of river and contaminated seepage water.     

Disparity between blood pressure and PRA inhibition after administration of a renin inhibitor to anesthetized dogs: methodological considerations

A dissociation between changes in blood pressure (BP) and plasma renin activity (PRA) has been noted after administration of renin inhibitors. In the present study, the renin inhibitor PD 132002 was given to salt-deplete, anesthetized dogs. PRA was measured at pH 6.0 by a conventional angiotensin I (ANG I) RIA method (PRA-C) and by an ANG I antibody-trapping RIA method (PRA-AT) performed at pH 7.4. PD 132002 at 0.01, 0.1, 1, and 10 mg/kg IV, reduced BP by 3 +/- 2, 9 +/- 2, 24 +/- 4, and 39 +/- 4 mm Hg, respectively, (baseline of 136 +/- 8 mm Hg, N = 5), when infused IV over 30 minutes with a 30 minute recovery between doses. The BP response at 10 mg/kg equaled that of saralasin (20 micrograms/kg/min IV). PRA-AT (baseline of 20 +/- 6 ng ANG l/ml/hr, N = 4) was inhibited by 0%, 28% +/- 12%, 75% +/- 10%, and 97% +/- 1% at 0.01, 0.1, 1, and 10 mg/kg, respectively. Plasma concentrations of immunoreactive ANG II were also reduced dose-dependently and paralleled changes in BP. In contrast, PRA-C (baseline of 13 +/- 4 ng ANG l/ml/hr, N = 4) was inhibited by 82% +/- 8% at 0.01 mg/kg and by > 98% at higher doses. After a single dose of PD 132002 at 10 mg/kg infused over 30 minutes, BP recovery paralleled changes in immunoreactive ANG II and PRA-AT, yet PRA-C inhibition showed no recovery over the same time course. Our data support the conclusion that BP relates better to PRA-AT than PRA-C. Thus the dissociation sometimes observed in studies with renin inhibitors between changes in BP and PRA may be attributed to the assay used to determine PRA.     

Application of ion-exchange cartridge clean-up in food analysis. I. Simultaneous determination of thiabendazole and imazalil in citrus fruit and banana using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible analytical method for thiabendazole (TBZ) and imazalil (IMA) in citrus fruit and banana has been developed. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of TBZ and IMA from citrus fruits spiked at levels of 10 microgram/g and 5 microgram/g were in the range of 94-98% and 93-98% with coefficients of variation of 0.5-2.2% and 1.6-2.7%, respectively. The recoveries of TBZ and IMA from banana spiked at levels of 3 microgram/g and 2 microgram/g were 94% and 94% with coefficients of variation 1.1% and 4.9%, respectively. The detection limits for TBZ and IMA were 0.1 microgram/g in citrus fruit and 0.05 microgram in banana.     

Growth hormone responses to growth hormone-releasing hormone and clonidine before and after erythropoietin therapy in CAPD patients

Correction of anemia with recombinant human erythropoietin (rhEPO) in patients with end-stage renal disease has been associated with improvement of several abnormalities in hypothalamo-hypophyseal functions. The aim of the present work was to evaluate the growth hormone (GH) responses to GH-releasing hormone (GHRH) and clonidine stimulation, as well as the baseline concentrations of insulin-like growth factor I(IGF-I), before and after the correction of anemia with rhEPO in a group of uremic patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Nine clinically stable patients (1 male, 8 female; mean age 55.4 years; mean duration of CAPD 14.1 months) were studied. Twelve normal volunteers were studied as controls. GHRH and clonidine stimulation tests were performed prior to starting rhEPO and again after partial correction of anemia with rhEPO therapy (60-130 U/kg/week, s.c., for 12 weeks). Blood samples for GH were collected during 2 h after GHRH (100 micrograms i.v. in bolus) or clonidine (0.15 mg/m2, p.o.) administration. In basal plasma samples IGF-I concentrations were also measured. Mean (+/- SEM) blood hemoglobin concentration rose from 5.32 +/- 0.25 to 7.22 +/- 0.25 mmol/l (p < 0.001) after rhEPO treatment. GH responses to GHRH were characterized by marked differences in single patients when compared with the control group. However, the GH peak and the area under the secretory curves (AUC) of GH responses in CAPD patients (9.89 +/- 4.01 micrograms/l and 15.06 +/- 6.02 micrograms.h/l, respectively) did not differ from those obtained in control subjects (14.58 +/- 3.25 microgram/l and 16.94 +/- 4.31 microgram.h/l, respectively). The study after correction of anemia showed an evident potentiation of GH values that reached statistically significant values at 60 and 90 min. GH AUC after rhEPO therapy rose to 25.61 +/- 9.25 micrograms.h/l (p = 0.01). In control subjects, clonidine administration was followed by a GH release that reached a maximum at 90 min (7.67 +/- 2.24 micrograms/l). However, CAPD patients exhibited a blunted response to clonidine both before (2.00 +/- 0.78 microgram/l) and after (2.78 +/- 0.76 microgram/l, NS) correction of the anemia with rhEPO. On the other hand, IGF-I concentrations after rhEPO therapy (32.05 +/- 5.52 nmol/l) were not significantly different from those found prior to starting therapy (38.13 +/- 8.44 nmol/l). In conclusion, these results suggest that correction of the anemia with rhEPO therapy potentiates GH responses to direct pituitary stimulation with GHRH although it is unable to restore the blunted response of GH to clonidine that is found in CAPD patients.     

Determination of pyridonecarboxylic acids in plasma by reverse-phase high-performance liquid chromatography

A simple qualitative and quantitative determination for pyridonecarboxylic acids including nalidixic acid (NA), oxolinic acid (OA) and pipemidic acid (PPA) in chicken plasma was carried out by microbiological, spectrophotometric, thin-layer chromatographic (TLC) and reverse-phase high-performance liquid chromatographic (HPLC) methods. As a test organism for bacteriological bioassay, Bacillus subtilis ATCC-6633 was the most sensitive of seven organisms investigated. Using the cup and the disc methods, a standard curve was obtained by determining the relationship between various drug concentrations and the diameter of the inhibition zone. The three drugs had two strong UV absorbance wavelengths (257 and 330 nm) on spectrophotometry. TLC analysis using a silica gel 60 F254 plate was investigated, and a solution of methanol:chloroform:acetic acid (3:1:1, v/v/v) was found to be the most suitable solvent for separation. The minimum concentration of drug detectable by this method was 0.5 microgram/ml for NA, 0.075 microgram/ml for OA and 0.39 microgram/ml for PPA. For HPLC analysis, a solution of acetonitrile:0.2 M phosphoric acid (1:1, v/v) was superior, and simultaneous determination of all three drugs was possible under the HPLC conditions used. The lowest measurable amount of drug in chicken plasma was 0.01 microgram/g. Recovery from extracts spiked with each drug at a known concentration was close to 100% for NA and OA, but only about 50% for PPA.