Cosegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood pressure.

The spontaneously hypertensive rat (SHR) exhibits alterations in the renin-angiotensin-aldosterone system which are similar to those that characterize patients with "nonmodulating" hypertension, a common and highly heritable form of essential hypertension. Accordingly, we determined whether the inheritance of a DNA restriction fragment length polymorphism (RFLP) marking the renin gene of the SHR was associated with greater blood pressure than inheritance of a RFLP marking the renin gene of a normotensive control rat. In an F2 population derived from inbred SHR and inbred normotensive Lewis rats, we found the blood pressure in rats that inherited a single SHR renin allele to be significantly greater than that in rats that inherited only the Lewis renin allele. To the extent that the SHR provides a suitable model of "nonmodulating" hypertension, these findings raise the possibility that a structural alteration in the renin gene, or a closely linked gene, may be a pathogenetic determinant of increased blood pressure in one of the most common forms of essential hypertension in humans.

Factors influencing the survival of rat brain tumor cells after in vitro treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The shape of dose-response curves obtained for asynchronous, exponentially growing 9L rat brain tumor cells treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea changed as a function of the drug exposure time. For short treatment times (less than 1 hr), the dose-response curves had shoulders, indicating that the cells may accumulate sublethal damage; however, after longer treatments (greater than 1 hr), little if any shoulder was apparent. The slope of the exponential portion of the dose-response curve increased progressively with treatment periods from 15 min to 2 hr. Longer exposure times (up to 24 hr) produced no further changes in the cell-kill kinetics. Cell survival was directly related to the BCNU exposure dose [o integral t Co(t)dt] and to the amount of bound BCNU per cell. Extrapolation of the curves for these two variables indicated that some BCNU damage accumulates before death occurs. The amount of serum and cell products available in the medium to bind BCNU affected the level of survival; however, there was no evidence that extracellular spontaneous breakdown products or chemical transformation products were involved in the cell-killing mechanism.

Urinary catecholamines in iron-deficient rats at rest and following surgical stress.

The purpose of this study was to determine catecholamine concentrations both at rest and in response to a surgical stress in iron-deficient and control rats. Twenty-one-day-old rats were randomized to one of two groups which received a diet containing either 6 or 50 mg iron/kg. Three to five days later, when anemia was first detectable, urinary norepinephrine (NE) concentrations were already significantly elevated in the iron-deficient compared to control rats. In contrast, urinary dopamine (DA) became depressed after 10 days of the iron-deficient regimen. At 38 days of age, both groups were subjected to a surgical stress. NE and DA became elevated over baseline values in both diet groups during the 24-h period following surgery; NE remained significantly higher and DA significantly lower in the iron-deficient than in the control group. We conclude that changes in urine catecholamine concentration occur early in the development of iron deficiency and that they are characteristic of both baseline and stress conditions.

Liquid chromatography with fluorometric detection of unconjugated estriol in serum of pregnant women.

We describe a fluorometric liquid chromatographic assay for the measurement of unconjugated estriol in the serum of pregnant women. Estriol is extracted into methylene chloride/propanol-2 from serum by use of a Clin-Elut extraction column, the extract evaporated, and the residue redissolved in mobile phase. An aliquot is injected onto the liquid chromatograph and the estriol is separated on a reversed-phase octyl column with a mobile phase consisting of acetonitrile/phosphate buffer (23:77, v/v). The effluent is monitored by fluorescence detection. The proposed method offers good reproducibility (CV less than 7%), sensitivity (less than 0.5 micrograms/l), and accuracy. Of many drugs and steroids tested, only 16,17-epiestriol interferes with the estriol analysis.

Simultaneous liquid chromatographic analysis of amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and nordoxepin.

A simultaneous method for the therapeutic monitoring of amitriptyline, doxepin, imipramine, and their active demethylated metabolites nortriptyline, nordoxepin, and desipramine, respectively, in plasma or serum by reversed-phase liquid chromatography (RPLC) is presented. The drugs and the internal standard (loxapine) are first extracted from 2 ml of serum into butylchloride at pH 14, and then back extracted into 200 microliter of 0.025 mol/l hydrochloric acid. An aliquot of the aqueous acid phase is injected into the chromatograph and eluted with acetonitrile-phosphate buffer (21: 79, by vol.) containing 0.6 nl of n-nonylamine per liter of phosphate buffer. The drugs are eluted in a total chromatographic time of approximately 13 min at ambient temperature and detected at 200 nm. A sensitivity of 5 microgram/l of serum for each drug is obtained. Recoveries for these drugs ranged from 77% to 103%; and the coefficient of variation (day-to-day) ranged from 4.2 to 7.8. Of 35 basic or neutral drugs tested for possible interference, only propoxyphene interferes with the analysis of nortriptyline.

Pharmacokinetics of intracarotid artery 14C-BCNU in the squirrel monkey.

A comparison of intravenous to intracarotid artery (ICA) administration of 14C-BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) was made in squirrel monkeys. Radioactivity was measured as soluble drug products and as RNA-, DNA-, and protein-bound radioactivity. The ICA administration of BCNU achieved 190% to 280% higher brain nucleic acid-bound drug levels than use of the intravenous route in the infused hemisphere and 130% to 280% higher levels than in the noninfused hemisphere. In addition, some brain regions directly subserved by the middle cerebral artery had bound drug levels four- to fivefold greater than those found in regions of noninfused brain. The data suggest that a need for BCNU dose reduction due to myelotoxicity may be an indication for ICA therapy in selected brain-tumour cases.

Simple analogs of the toxin callicarpone.

Callicarpone, a component 10 times as toxic to fish as rotenone, has been isolated from the leaves of Callicarpa candicans. It is reasonable to assume that callicarpone will act as an insecticidal agent as does rotenone. Therefore, the structure-activity relationship of callicarpone was examined by synthesizing a series of compounds having certain of its structural features. Those compounds were tested for insecticidal and antimicrobial activities. A study of synthetic analogs elucidated the functional group chemistry of callicarpone so that a synthesis might be undertaken. Piperitone oxide showed approximately 1/100th the activity of rotenone against Daphnia magna. 1-(alpha-Hydroxyisopropyl)-3-oxocyclohexene oxide showed activity against myobacterium while 2,3,4,6,7,8-hexahydronaphthalene-1,4-dione showed inhibitory activity against the mycobacterium and two yeasts.

Liquid chromatographic determination of gentamicin in serum with spectrophotometric detection.

A simple, accurate, and specific liquid chromatographic procedure is described for the determination of gentamicin in 50 muL of serum. Gentamicin and sisomicin (IS) were converted into their trinitrophenyl derivatives by reaction with a water soluble derivatizing agent at 70 degrees C for 30 minutes. The derivatives were extracted from the crude mixture with chloroform, which was separated, evaporated, and the residue reconstituted in acetonitrile. Gentamicin was separated into three major isomers (C1, C2, and C1a) on a reversed-phase octyl column by eluting with a mobile phase. The eluted compounds were detected at 340 nm and quantified from their peak heights or peak areas. Chromatography was complete in less than 11.0 minutes. The lower limit of detection for gentamicin was less than 0.5 mg/L, analytical recoveries varied from 96.6% to 99.3%, linearity extended to 15.0 mg/L, and day-to-day precision was between 2.2% to 2.9%.

Rapid method for screening toxic drugs in serum with liquid chromatography.

We present a method for the simultaneous analysis of a variety of commonly abused drugs (acetaminophen, theophylline, salicylate, primidone, methyprylon, phenobarbital, butabarbital, ethchlorvynol, butalbital, chlordiazepoxide, pentobarbital, amobarbital, phenytoin, glutethimide, secobarbital, flurazepam, nitrazepam, methaqualone, N-desmethyldiazepam, and diazepam) in serum or plasma. Serum proteins are precipitated with an acetonitrile solution containing hexobarbital, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of acetonitrile/phosphate buffer (pH 3.2), using a two-step linear gradient, at a flow rate of 3.0 mL/min. The eluted drugs are detected by their absorption at 210 nm; their quantities are estimated from their peak heights. A complete analysis requires no longer than 45 minutes at the optimum column temperature of 50 degree C. A sensitivity of 2 mg/L of serum is attained routinely for most of the hypnotic and analgesic drugs; while methaqualone, chlordiazepoxide, diazepam, and N-desmethyldiazepam can be detected at a concentration of 0.2 mg/L. Analytical recoveries for the twenty drugs varied from 93-112%, with good reproducibility. Of more than forty drugs tested for possible interference, desmethyldoxepin, procainamide, phenylpropanolamine, mesantoin, and phenacetin interfere with the analysis of flurazepam, acetaminophen, ethchlorvynol, and phenobarbital, respectively.

Clinical analysis of individual steroids by column liquid chromatography.

At present, there are various LC methods available for the determination of estriol, aldosterone, and cortisol in urine, and for cortisol, cortisone, 11-deoxycortisol, corticosterone, 17-hydroxyprogesterone, estriol, vitamin D isomers, and various exogenous glucocorticoids in serum. The LC methods are more specific than the currently available RIAs or homogenous immunoassays. However, whether the data obtained by more specific LC methods are better clinically than the commonly used immunoassays for these steroids must still be proven. In this review, I have critically evaluated various LC methods currently available for the routine determination of clinically important steroids in the clinical laboratories. A complete evaluation and advantages and disadvantages of alternative techniques are beyond the scope of this review. However, the readers are urged to refer to the review articles and chapters listed in the reference section.

Extended life for blood serum analysis columns using dual zone chromatographic materials.

Column lifetimes of novel dual zone material (DZM) adsorbents were evaluated by direct serum injection analysis for drugs by high-performance liquid chromatography. Porous silica was converted to DZMs in which the outer zone was enriched in an immobilized lipophobic moiety, the perfluorobutylethylenesilyl group, and the internal zone was enriched in a lipophilic octadecylsilyl group. Performance during repetitive serum analyses for phenobarbital and carbamezepine by reverse phase HPLC was compared to that of control adsorbents of the same composition but without the dual zone distribution. The DZM columns had lifetimes up to 4 times longer than the controls. Furthermore, under these conditions, even the control columns had considerably longer lifetimes than conventional but more retentive reverse phase columns. The DZM column lifetimes also appeared to be comparable to or better than those of the recently introduced Pinkerton packings and have much better adsorbent design flexibility and pH operating latitute.

Liquid chromatographic analysis of clonazepam in human serum with solid-phase (Bond-Elut) extraction.

A simple, sensitive, selective and precise liquid-column chromatographic assay for clonazepam is described, in which 1 ml of serum containing 50 micrograms/l methylclonazepam as an internal standard is extracted by elution from a Bond-Elut column with 400 microliter of methanol. An aliquot of the eluate is injected on to a reversed-phase column and eluted with a mobile phase of acetonitrile--phosphate buffer (30:70) at a flow-rate of 2 ml/min at a column temperature of 50 degrees C. Detection is at 254 nm. Chromatography is complete in 12 min. A sensitivity of 2 ng/ml is attained when 1 ml of serum is extracted. Analytical recovery of the clonazepam added to serum ranged from 91% to 99% with a coefficient of variation of 6.0%. This assay for clonazepam has good precision, with coefficients of variation of 11% at 15 ng/ml and 2.6% at 50 ng/ml. There was no interference from any of the commonly used antiepileptics.

High-pressure liquid-chromatographic determination of 5-(4-hydroxyphenyl)-5-phenylhydantoin in humanurine.

We describe a sensitive and precise high-pressure liquid-chromatographic method for measurement of total 5-(4-hydroxyphenyl)-5-phenylhydantoin, a metabolite of phenytoin, in urine. An aliquot of urine, containing 5-(4-methylphenyl)-5-phenylhydantoin as an internal standard, is processed and chromatographed. The metabolite and internal standard are identified from their retention times and quantitated from their relative response factors. The metabolite is separated from normal urine constituents and internal standard in less than 8 min. The sensitivity of the method is such that after the usual dose it can be measured in 0.5 ml of urine; the lower limit of detection is 300 ng.

Determination of carbamazepine in blood or plasma by high-pressure liquid chromatography.

We described a sensitive and precise high-pressure liquid-chromatographic method in which 5-(p-methylphenyl)-5-phenylhydantoin is used as the internal standard in determining carbamazepine in whole blood or plasma. Carbamazepine is well separated from normal blood constituents in less than 8 min, and other commonly used anticonvulsants do not interfere with the analysis. The sensitivity of this method is adequate to quantitate 0.25 mg of carbamazepine per liter in 2 ml of sample, and the lower limit of detection is 100 ng. Twenty specimens were analyzed by a gas-chromatographic method and by the present method; the resulting correlation coefficient was greater than .980.

Liquid-chromatographic analysis for serum theophylline in less than 70 seconds.

We describe a sensitive, specific, and very fast liquid-chromatographic assay for serum theophylline, involving a commercially available high-speed reversed-phase column and a micro-flow-cell-equipped detector. Each analysis requires only 100 microL of serum (as little as 25 microL may be used when necessary), and chromatography is complete in less than 70 s. Analytical recovery of theophylline added to serum ranged from 97 to 102%. Between-run precision (CV) ranged from 2.1 to 3.5%. The lower limit of detection for theophylline is 0.5 mg/L, and linearity extends to 50 mg/L. Numerous drugs and xanthine metabolites tested do not interfere.

Liquid chromatographic determination of cyclosporine in whole blood with the advanced automated sample processing unit.

We describe a rapid, precise, cost-effective, and accurate isocratic liquid chromatographic (LC) procedure for determining cyclosporine in whole blood. The cyclosporine is extracted from 0.5 ml of whole blood together with 200 micrograms of cyclosporin D, added per liter as internal standard, by using an Advanced Automated Sample Processing (AASP) unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge which is interfaced with a Perkin-Elmer 83 X 4.6 mm I.D. cartridge column, packed with 3-micron octadecyl packing. The column is eluted with a mobile phase containing acetonitrile-water (13:7) at a flow-rate of 1.0 ml/min at a column temperature of 70 degrees C. The column effluent is monitored at 210 nm. The absolute recovery of cyclosporine exceeded 87% and the linearity extended up to 2000 micrograms/l. Within-run and day-to-day coefficients of variation were less than 8%. The correlation between AASP-LC and manual Bond-Elut extraction-LC method was excellent (r = 0.97).

Simultaneous very fast liquid-chromatographic analysis of ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine in serum.

We describe a sensitive, specific, and very fast liquid-chromatographic assay for simultaneously determining five anticonvulsants (ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine) by using commercially available 5- or 3-microns particle size reversed-phase columns and a microflow-cell-equipped ultraviolet detector. The anticonvulsant drugs are extracted from 200 microL of serum containing 50 mg of cyclopal per liter as an internal standard, by elution from a Bond-Elut (Analytichem International, Harbor City, CA 90710) column with 300 microL of methanol. A 5-microL aliquot of the eluate is applied to an analytical column and eluted with a mobile phase of acetonitrile/methanol/phosphate buffer, 20 mmol/L, pH 3.7 (13.5/35/51.5 by vol), at a flow rate of 3.0 mL/min and at 50 degrees C. Detection is at 210 or 195 nm. The chromatography is complete in less than 2.5 min with the 5-microns-particle column, and in less than 1.4 min with the 3-microns-particle column. The sensitivity of the method for all drugs is less than 1 mg/L. Analytical recovery of drugs added to serum ranged from 92 to 109% for concentrations up to 200 mg/L. Between-run precision (CV) ranged from 1.3 to 4.1%.

Automated solid-phase extraction and liquid chromatography for assay of cyclosporine in whole blood.

In this rapid, precise, accurate, cost-effective, automated liquid-chromatographic procedure for determining cyclosporine in whole blood, the cyclosporine is extracted from 0.5 mL of whole blood together with 300 micrograms of cyclosporin D per liter, added as internal standard, by using an Advanced Automated Sample Processing unit. The on-line solid-phase extraction is performed on an octasilane sorbent cartridge, which is interfaced with a RP-8 guard column and an octyl analytical column, packed with 5-microns packing material. Both columns are eluted with a mobile phase containing acetonitrile/methanol/water (53/20/27 by vol) at a flow rate of 1.5 mL/min and column temperature of 70 degrees C. Absolute recovery of cyclosporine exceeded 85% and the standard curve was linear to 5000 micrograms/L. Within-run and day-to-day CVs were less than 8%. Correlation between automated and manual Bond-Elut extraction methods was excellent (r = 0.987). None of 18 drugs and four steroids tested interfered.

Solid-phase extraction and liquid chromatography for improved assay of cyclosporine in whole blood or plasma.

In this simple, precise, accurate, and specific isocratic liquid chromatographic procedure for determining cyclosporine, the cyclosporine is extracted from 1 mL of whole blood or from plasma, with 500 micrograms of cyclosporin D added per liter as internal standard, by elution from a Bond-ElutTM C18 extraction column with 300 microL of a mixture of ethanol and tetrahydrofuran. A 100-microL aliquot of the eluate, injected onto a cyano-phase analytical column, is eluted with a mixture of acetonitrile and pH 7.0 phosphate buffer at a flow rate of 1.0 mL/min and at 50 degrees C. Detection is at 210 nm. The chromatography is complete in less than 14.0 min. The method can measure less than 10.0 micrograms/L. Analytical recovery of cyclosporine added to whole blood ranged from 99 to 109% for concentrations up to 2000 micrograms/L. Between-run CVs ranged from 6.4 to 6.6%. None of numerous drugs and steroids tested interfered. Results by radioimmunoassay exceeded by 20 to 350% those measured by the present method.

Improved liquid-chromatographic method for determination of serum cortisol.

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.