Polarographic method for rapid microdetermination of cholesterol with cholesterol esterase and cholesterol oxidase.

Cholesterol concentrations in serum are enzymatically determined rapidly by use of a polarographic oxygen analyzer with a circuit modified to record simultaneously the amount and rate of oxygen consumption. The final assay system, assessed from the oxygen consumption value that we found to be optimum, consists of 1 ml of sodium phosphate buffer (0.6 mol/liter, pH 7.0) containing NaN3 (10 mg/liter), Triton X-100 surfactant (10 ml/liter), 0.4 U of cholesterol ester hydrolase, and 0.6 U of cholesterol oxidase. Oxygen consumption and cholesterol concentration are linearly related to 8.0 g/liter, and only 10 mul of serum is required. Replicate analyses of pooled serum by the present method demonstrated the following inter-run precision: mean = 1731 mg/liter, SD = 22.3 mg/liter, CV = 1.3%. Bilirubin and ascorbic acid were without effect on the present method, unlike the enzymatic colorimetric methods.     

Gas chromatographic/thin-layer chromatographic analysis of acetylated codeine and morphine in urine.

This procedure positively identifies codeine and morphine in urine. Urine samples are hydrolyzed and extracted with organic solvent, and the extracts are evaporated and acetylated. The presence of codeine and morphine is ascertained by gas chromatography (3% OV-25 and 3% Poly-A 103 columns) and confirmed by thin-layer chromatography (system: ethyl acetate/acetone/concd ammonium hydroxide, 100/10/4.5 by vol; reagent: iodoplatinate). As little as 0.5 mg each of codeine and morphine per liter, in free and conjugated forms, is detectable by this method.     

Therapeutic monitoring of anticonvulsant drugs: gas-chromatographic simultaneous determination of primidone, phenylethylmalonamide, carbamazepine, and diphenylhydantoin.

We describe a sensitive and precise gas-chromatographic method in which benzylmalonate methylester monoamide is used as the internal standard for the simultaneous determination of primidone, phenylethylmalonamide, carbamazepine, and diphenylhydantoin. The trimethylsilyl derivatives of the anticonvulsants are well separated from each other and from normal serum constituents. The lower limit of detection for each drug is 0.5 mg/liter when 1 ml of serum is analyzed. Within-run precision (CV), established by analysis of 10 replicates, was as follows: primidone (5.4 mg/liter), 2.6%; phenylethylmalonamide (5.5 mg/liter), diphenylhydantoin (6.6 mg/liter), 3.8%; and carbamazepine (10.4 mg/liter), 3.2%. Fifty specimens were analyzed for primidone and 35 for diphenylhydantoin by a standard gas-chromatographic method involving on-column methylation and by the procedure we have developed. The mean value observed for primidone with the on-column alkylation procedure was 9.3 mg/liter and with our procedure was 9.6 mg/liter. When values for our assay were regressed against values for the standard method, the slope of the least-squares line was 0.936, the intercept was 1.00 mg/liter, and r was 0.939. The mean values observed for diphenylhydantoin by on-column methylation and with our procedure were both 12.6 mg/liter. When values for our assay were regressed against the standard method, the slope of the least-squares line was 0.944, the intercept was 0.3 mg/liter, and r was 0.988.     

Determination of creatinine in serum and urine by a rapid liquid-chromatographic method

We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.     

Gas-chromatographic quantitation of theophylline in small volumes of plasma.

We describe a rapid procedure for quantitating theophylline in 100-mul plasma samples by use of a gas-liquid chromatograph equipped with a flame ionization detector. This methos is especially useful for monitoring theophylline concentrations in serum or plasma of infants, because sufficiently large blood samples can be readily obtained from a heel prick. The method is specific for theophylline in the presence of caffeine, theobromine and phenobarbital. For plasma concentrations equal to or greater than 5 mg/liter the average daily coefficient of variation was less than 7% while the coefficient of variation from day to day was less than 11%. The same approach can also be used to measure concentrations of phenobarbital in small volumes of plasma or serum, and is readily adapted to determination of theophylline and phenobarbital in larger samples.     

Improved colorimetric determination of salicylic acid and its metabolites in urine.

We describe an improvement in the Levy and Procknal method [J. Pharm. Sci. 57, 1330 (1968)] for determination of salicylic acid and its metabolites in urine. Salicylic acid and salicyluric acid are successively extracted from 1 or 2 ml of urine (acidified with HCl) by two 10-ml portions each of carbon tetrachloride and ethylene dichloride. The extracts of each solvent are shaken with 5 ml of ferric nitrate solution (a 10-fold dilution of 17 g of Fe(NO-3)-9H-2O in 1 liter of 70 mmol/liter HNO-3). The aqueous phases are centrifuged and their absorbances measured at 530 nm. For total salicylate, 3 ml of urine and 3 ml of HCl are heated in a partially evacuated serum vial at 100 degrees C for 16 h and then salicylic acid is assayed in the hydrolyzed sample. Recovery of a weighed oral dose of sodium salicylate in urine was 105.4%; it was 127.9% by the Levy and Procknal method for the same sample. The improved method is faster and more accurate.     

Effects of standard breakfast on pharmacokinetics of oral zidovudine in patients with AIDS.

The influence of a standard breakfast on the single-dose pharmacokinetics of zidovudine (AZT) after oral administration of 100 and 250 mg of AZT was studied in 27 subjects with advanced human immunodeficiency virus infection (Centers for Disease Control stage IV). Concentrations of AZT and the 5'-glucuronide metabolite (GAZT) in serum and urine were measured by a high-pressure liquid chromatographic method. Pharmacokinetic analysis was done by an open one-compartment model as well as noncompartmentally. The results were summarized as medians with 50% confidence ranges because of the high degree of interindividual variability. Peak levels in plasma were moderately reduced after administration of 100 mg AZT in the nonfasting group (1.79 mumol/liter in the fasting group [F], 1.12 mumol/liter in the group that received breakfast [B]) and were markedly reduced after administration of 250 mg AZT (6.51 mumol/liter [F], 1.79 mumol/liter [B]). The terminal half-life in plasma was prolonged almost twofold after breakfast with 100 and 250 mg of AZT (100 mg, 36.4 min [F] and 51.6 min [B]; 250 mg, 35.3 min [F] and 63.6 min [B]). Recoveries (AZT and GAZT) in urine varied with both dosages, reflecting more a problem of accounting for the metabolite GAZT in urine than a relevant difference (100 mg, 115% [F] and 76.5% [B]; 250 mg, 71% [F] and 99.4% [B]). Our data suggest that absorption of AZT in human immunodeficiency virus-infected subjects is extremely variable, with a high degree of interindividual differences. Furthermore, breakfast had a marked influence on the absorption of AZT, suggesting that the drug should be taken in a fasting state.     

Direct determination of inorganic phosphor in serum and urine utilizing Rhodamin B (author's transl)]

A micromethod is described for the spectrophotometric determination of inorganic phosphate in serum and urine utilizing Rhodamin B as a dye (phosphomolybdat-Rhodamin B complex), Brij 35, and polyvinylpyrrolidon as catalyst. The procedure does not involve deproteinization and yields a stable complex in 20 min. The linearity is constant up to at least 9 mg Pi per 100 ml. It is both accurate (r = 0.972 in serum, r = 0.989 in urine, recovery in urine 98%) and precise (vk = 1.16% in the series). Bilirubin up to 10 mg/100 ml and serum protein do not interfere with the method.     

Simultaneous detection of morphine and barbiturates in urine by radioimmunoassay.

This report describes a radioimmunoassay for the simultaneous detection of morphine and barbiturates. Morphine and barbiturate antibodies, obtained from goats, were mixed with 125l-labeled antigens. By adjusting concentrations of the morphine and barbiturate antibodies and radiolabeled antigens, closely superimposed standard curves for the two drugs would be obtained. As a consequence, similar response curves were obtained for urine specimens containing morphine or barbiturates. Although concentrations as low as 25 mug/liter could be measured, to ensure against false positive reactions the test should be performed at the 100 mug/liter concentration. Unknown samples positive by the dual assay were confirmed by separately testing the specimens with the individual radioimmunoassay specific for morphine or barbiturate. Equivalency tests of urines positive for morphine, positive for barbiturates, or negative for both demonstrated complete correlation between the single and dual assays. The mixed reagent retained its sensitivity and specificity for at least three months when stored at 4 or 25 degrees C. The dual radioimmunoassay is a rapid, simple procedure that can be adapted to automated processes and that is suitable for large- and small-scale screening.     

Liquid-chromatographic separation of urinary 5-hydroxy-3-indoleacetic acid, with measurement at 254 nm

A "high-performance" liquid-chromatographic procedure for 5-hydroxy-3-indoleacetic acid is described and compared with a colorimetric method in which 1-nitroso-2-naphthol is used. The analyte and an internal standard, p-nitrobenzoic acid, were extracted into diethyl ether from urine at pH 4.0 (acidified with HCl) to which sodium chloride had been added, and the ether was back-extracted with acetate buffer, pH 9.2. Aliquots of this extract were injected into a reversed-phase liquid-chromatographic column and eluted with pH 3.5 acetate buffer/methanol (95/5 by vol); the effluent was monitored at 254 nm. The precision (CV) of the method was 11.8% at 1.8 mg/L, 5.5% at 92 mg/L. Analytical recovery averaged 84%. The colorimetric method gave higher values for the analyte than did the chromatographic method for all patients' urines.     

An improved micro-scale liquid-chromatographic assay for piperacillin in plasma and urine

An accurate, sensitive, and specific liquid-chromatographic method is described for measuring piperacillin in plasma and urine. Plasma samples deproteinized with two volumes of acetonitrile containing 1.2 mg of the internal standard, p-nitrobenzene sulfonamide, per liter are centrifuged. The clear supernate is evaporated under nitrogen, and the residue is reconstituted in 50 microL of the mobile phase (32/68 by vol acetonitrile/water, adjusted to pH 2.5 with 85% phosphoric acid), of which 10 microL is injected onto a reversed-phase (C-18) column. Urine samples are diluted 10-fold with distilled water, an equal volume of acetonitrile containing 3 mg of the internal standard per liter is added, and 20 microL is chromatographed. Stability studies indicate that storage conditions are critical for both plasma and urine. Piperacillin in plasma is stable at -70 degrees C for at least six weeks, but 100% of it is degraded during the same time at -20 degrees C. Piperacillin in urine is also stable at -70 degrees C for six weeks, but 20% is degraded during six weeks at -20 degrees C.     

Kinetic enzymatic method for determining serum creatinine.

Creatinine amidohydrolase is used to measure serum creatinine in a totally enzymatic procedure. Creatine, produced by hydrolysis, is acted upon by creatine kinase, and then by pyruvate kinase and lactate dehydrogenase, to result in a change in absorbance at 340 nm. The amount of creatinine present is related to the rate of change in A340 and is determined from a standard curve. Absorbance and concentration are linearly related to 100 mg/liter and only 250 mul of serum is required. At 1.0 g/liter, heparin, oxalate, citrate, ethylenediaminetetraacetate, ascorbate, or glucose had no significant effect on the accurate determination of creatinine; higher concentrations (30 g/liter) had inhibitory effects on the test. Analytical recovery of creatinine added to either normal or abnormal sera averaged 102%. When results of this procedure and of the standard direct Jaffé test were compared, the latter were significantly higher. Unlike the Jaffé method, the present method of determining creatinine is rapid (about 10 min per test), subject to few or no interfering substances, and requires no serum deproteinization.     

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).     

Simultaneous measurement of phenobarbital, phenytoin, primidone, ethosuximide, and carbamazepine in serum by high-pressure liquid chromatography.

We present a method for simultaneously determining five anticonvulsants [phenobarbital, phenytoin (diphenylhydantoin), primidone, ethosuximide, and carbamazepine] in as little as 25 microliters of serum. The proteins are precipitated with an acetonitrile solution containing hexobarbital as an internal standard. The anticonvulsants are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer (19/81 by vol) at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm, and quantities estimated from their peak heights. Each analysis requires about 14 min. at an optimum column temperature of 50 degrees C. The lower unit of detection for all of these drugs is less than 10 ng. Sensitivities, for serum samples, of 1.0 mg/liter for all the drugs analyzed are attained routinely. Analytical recoveries for the five drugs varied from 97 - 107%, with good day-to-day precision (CV between 3.9 and 5.9%). Of more than 30 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.     

应用连二亚硫酸钠判断百草枯中毒的程度和预后

目的 应用连二亚硫酸钠显色法判断百草枯中毒的程度和预后.方法 将不同浓度的百草枯标准品与过量连二亚硫酸钠在碱性条件下反应,建立百草枯检测标准比色卡和标准曲线.应用连二亚硫酸钠显色法前瞻性检测浙江大学医学院附属第一医院2008年1月至2009年5月间收治的22例百草枯中毒患者血、尿液百草枯浓度,回顾性分析了患者就诊即刻血、尿百草枯浓度与患者预后的相关性.结果22例百草枯中毒患者经血液灌流、血浆置换和血液滤过治疗后随访3月以上,存活6例(27.3%).6例存活者就诊即刻百草枯半定量检测显示为极浅(3例)或浅(3例),16例死亡患者就诊即刻百草枯半定量检测显示为浅(1例)、中(2例)、深(2例)、极深(11例).6例存活患者与16例死亡患者就诊即刻尿百草枯质量浓度分别为(1.95±1.76)mg/L和(53.4±45.9)mg/L,P＜0.01.6例存活患者和13例死亡患者就诊即刻血清百草枯质量浓度分别为(1.70±1.39)ng/L和(29.5±22.1)mg/L,P＜0.01.22例百草枯中毒患者存活状况与就诊即刻尿百草枯半定量、定量检测结果 及血清百草枯质量浓度均显著相关(r_s分别为-0.804、-0.772,和-0.593,P＜0.01).结论 应用连二亚硫酸钠可迅速检测患者血、尿百草枯质量浓度,有助于了解患者中毒程度和判断预后.     

Assay for cyclo-, seco- and pentobarbital by multiple ion detection: kinetics after a single dose

A sensitive assay for the determination of four different barbiturates: butabarbital, cyclobarbital, pentobarbital and secobarbital is described. The barbiturates are determined quantitatively by gas chromatography/mass spectrometry (GC/MS) as their dimethylated derivatives. The sensitivity of this simple procedure is limited to 10 x 10(-6) g/l for cyclo- and secobarbital and 1 x 10(-6) g/l for pentobarbital in plasma. The assay has been used to measure the kinetics of pentobarbital over 6 days after a single 50 mg dose of the sodium salt, and over 3 days for one combined preparation of 25 mg cyclobarbital-calcium and 75 mg secobarbital-sodium. The corresponding barbiturate levels in saliva were determined by the same assay.     

Enzyme immunoassays with the miniature centrifugal fast analyzer.

We studied the EMIT (Enzyme Multiplied Immunoassay Technique, Syva) procedures for the assay of phenytoin and phenobarbital in serum, adapting them to the miniature Centrifugal Fast Analyzer. For different concentrations of drug, each rate of reaction decreased continuously with time, tending to converge on a single common value. The rate was most affected by the concentration of drug almost immediately after the reagents were mixed, less so thereafter. The antibody evidently is present in sufficient excess to bind all the enzyme-labeled drug ordinarily present, but the antibody-bound enzyme was only 75% inhibited; this helps explain the appreciable residual activity when no drug is present. The reaction course was the same whether the serum and enzyme-labeled drug were added to the antibody sequentially or simultaneously, which suggests that antibody is bound to drug appreciably faster than to enzyme-labeled drug. The reaction rates 15 to 30 s after mixing were used as the measure of the drug concentrations. These results were confirmed by noting the rates at successive 15-s intervals. The analyzer yielded a run-to-run CV of 10% for phenobarbital at 30 mg/liter, and 9% for phenytoin at 15 mg/liter, as compared to the 15% quoted by Syva.     

Acute sensitivity of FAST and SLOW mice to the effects of abused drugs on locomotor activity.

The universal nature of the stimulant or euphoric effect of addictive drugs suggests that it may be an important predictor of a drug's addiction potential. Furthermore, assessment of stimulant sensitivity could be useful for predicting the liability of individuals to drug abuse. The stimulant actions of abused drugs from different pharmacological classes may share a common biological mechanism. We investigated this notion by assessing the drug responses relative to base-line locomotor activity of mice selectively bred for increased (FAST) and reduced (SLOW) sensitivity to ethanol-induced stimulation. FAST mice were more sensitive than SLOW mice to the stimulant effects of methanol (1.5-3.0 g/kg), t-butanol (0.2-0.6 g/kg), n-propanol (0.15-1.2 g/kg), pentobarbital (10-40 mg/kg) and phenobarbital (15-120 mg/kg). FAST and SLOW mice were similarly stimulated by d-amphetamine (1.25-10 mg/kg) and caffeine (2.5-20 mg/kg). The activity of FAST and SLOW mice was equally depressed by nicotine (0.5-2.0 mg/kg) and morphine (4-75 mg/kg). Finally, FAST mice were unaffected, whereas SLOW mice were depressed by diazepam (1-8 mg/kg). Selection for relative sensitivity to stimulation by ethanol has generalized to other alcohols and to barbiturates, but not to several other abused drugs, including amphetamine. The data presented here support a hypothesized common mechanism of stimulant action for alcohols and barbiturates, and suggest that differences in sensitivity to drug stimulant effects can be seen in the absence of dopamine system differences.     

Concentrations in plasma, urinary excretion, and bactericidal activity of linezolid (600 milligrams) versus those of ciprofloxacin (500 milligrams) in healthy volunteers receiving a single oral dose

In a randomized crossover study, 12 volunteers (6 males, 6 females) received a single oral dose of 600 mg of linezolid or 500 mg of ciprofloxacin to assess the concentrations in plasma (up to 24 h), urinary excretion (by high-pressure liquid chromatography), and bactericidal titers in urine (UBT) at intervals up to 120 h. The mean maximum concentration of linezolid in plasma was 13.1 mg/liter, and that of ciprofloxacin was 2.46 mg/liter. The median cumulative levels of renal excretion of the administered dose of the parent drug were 44% for linezolid (range, 28 to 47%; mean +/- standard deviation, 40% +/- 7.8%) and 43% for ciprofloxacin (range, 20 to 56%; mean +/- standard deviation, 40% +/- 9.3%). The UBTs, i.e., the highest twofold dilution (with antibiotic-free urine used as the diluent) of urine that was still bactericidal, were determined for a reference strain and five gram-positive clinical uropathogens for which the MICs of linezolid and ciprofloxacin were as follows: Staphylococcus aureus ATCC 27278, 2 and 0.25 mg/liter, respectively; Staphylococcus aureus (methicillin susceptible), 1 and 16 mg/liter, respectively; Staphylococcus aureus (methicillin resistant), 2 and 64 mg/liter, respectively; Staphylococcus saprophyticus (methicillin susceptible), 1 and 0.25 mg/liter, respectively; Enterococcus faecalis, 2 and 1 mg/liter, respectively; and Enterococcus faecium, 2 and 1 mg/liter, respectively. The median UBTs of linezolid measured within the first 6 h were 1:96 for each of the two enterococcal strains and between 1:128 and 1:256 for the four staphylococcal strains. The median UBTs of ciprofloxacin were 1:64 for the two enterococcal strains; between 1:384 and 1:512 for the two ciprofloxacin-susceptible strains; and 1 (bactericidal activity of undiluted urine only) and 1:2 for the two resistant staphylococcal strains, respectively. The areas under the UBT-time curve (AUBT) for linezolid and ciprofloxacin showed no statistically significant (P<0.05) differences except for a better AUBT for linezolid for the two ciprofloxacin-resistant staphylococcal strains. For linezolid there were no statistically significant differences in UBTs or AUBTs for ciprofloxacin-susceptible and -resistant strains. Thus, the bactericidal activities of linezolid and ciprofloxacin against susceptible strains in urine were comparable, whereas linezolid also exhibited the same good bactericidal activity against ciprofloxacin-resistant strains. Therefore, linezolid should be tested for use as empirical treatment for complicated urinary tract infections due to gram-positive uropathogens in an appropriate clinical trial.     

Rapid high-pressure liquid chromatographic method for analysis of phenoxymethylpenicillin in human serum.

A selective high-pressure liquid chromatographic method for the determination of phenoxymethylpenicillin in human serum is described. The technique is based on the single extraction of the drug from acidified serum with diethyl ether. Chloramphenicol is used as internal standard. The chromatographic system consists of a reversed-phase C-18 column; the mobile phase is acetonitrile-0.01 M potassium acetate buffer (20:80 [vol/vol]; pH 6.5). The method can accurately measure serum penicillin concentrations down to 30 micrograms/liter with 500 microliter of sample. The coefficient of variation for intraassay variability of penicillin is between 1.5 and 4.9% in the range of 0.125 to 16.00 mg/liter. The extraction efficiency is 78.5 +/- 6.8% (+/- standard deviation; n = 9), and the calibration graph is linear in the concentration range studied. Pharmacokinetic data, obtained with the present method from seven healthy volunteers, are presented.