Reversed-phase LC assay method for deoxycholate in influenza vaccine.

Sodium deoxycholate is used for the disruption of particles in the manufacturing of some influenza vaccines. Residual deoxycholate in inactivated vaccines is currently determined using a labour-intensive colorimetric method which lacks complete specificity. An alternative assay method for residual deoxycholate in vaccine preparations was developed using reversed-phase LC. Cholic acid was used as internal standard and the ratio of internal standard to test solute was used for all calculations. Prior to LC analysis, deoxycholic acid was concentrated by solid-phase extraction, a procedure that also removed proteinaceous material in vaccine samples. The clean-up/concentration procedure recovery was examined using untreated samples and was found to be quantitative. The linearity range of the LC method was between 3 and 200 micrograms ml-1, with a limit of detection of approximately 0.4 micrograms on column, and a lower limit of quantitation of 1.6 micrograms on column. Replicate assays during intra-and inter-day experiments gave acceptable levels of variability. The DCA content of samples from three lots of influenza vaccine varied between 10 and 16 micrograms ml-1. These values were appreciably lower than those measured spectrophotometrically, indicating the higher specificity of the LC method.

Metabolism and disposition of phenazopyridine in rat.

1. The blood profile, tissue distribution, biliary and urinary excretion, and metabolism of 14C-phenazopyridine (PAP) was studied in male Wistar rats. 2. Based on the blood profile of 14C the absorption of PAP from the gastrointestinal tract was rapid; the t1/2 of elimination was 7.35 h. 3. Biliary excretion was a major route of elimination with 40.7% dose excreted by this route in bile duct-cannulated rats over the 0-8 h period. The predominant metabolite was conjugated 4'-hydroxy-PAP. 4. Liver and kidney showed the highest tissue levels of PAP-derived 14C, and significant covalent binding was found in these two tissues. 5. The major urinary metabolite of PAP was 4-acetylaminophenol (NAPA) followed in order by 5,4'-dihydroxy-PAP, 5-hydroxy-PAP, 4'-hydroxy-PAP and 2'-hydroxy-PAP; unchanged PAP accounted for < 1% dose. 6. Doubling the dose of PAP to 200 mg/kg caused a proportionate decrease in urinary NAPA excretion and an increase in 5-hydroxy-PAP.

Excretion of phenazopyridine and its metabolites in the urine of humans, rats, mice, and guinea pigs.

The metabolism of the urinary tract analgesic phenazopyridine [2,6-diamino-3-(phenylazo)pyridine; PAP] was studied in the urine of humans, rats, mice, and guinea pigs. Urinary excretion was rapid in human and guinea pig, but in the rat and mouse it was slower and there was significant fecal excretion. Metabolism of PAP was extensive in all four species, and there were marked quantitative differences in the routes of metabolism. The extent of azo bond cleavage was high in the mouse and guinea pig, moderate in the rat, and low in humans. Hydroxylation of both the phenyl and pyridyl rings of PAP was observed in all species. In the human, 5-hydroxyl PAP was the major metabolite (48.3% of the dose). It was concluded that there are marked species differences in the metabolism of PAP, and that none of the species studied resembles the human; the rat comes closest, but cannot be considered a particularly good model.

Postabsorption antidotal effects of N-acetylcysteine on acetaminophen-induced hepatotoxicity in the mouse.

Male Swiss Webster mice, treated with N-acetylcysteine (NAC, 500 mg/kg po) 1 h following acetaminophen (NAPA, 350 mg/kg po) administration, had control levels of transaminases indicating that NAC protects against NAPA-induced hepatotoxicity by postabsorption antidotal mechanism(s). Hepatic congestion induced by NAPA was reduced by NAC. Significantly higher elimination rate constants (K) for indocyanine green (500 micrograms/kg, iv) in mice treated with NAPA and NAC (K = 0.676 +/- 0.062) than in animals receiving NAPA alone (0.341 +/- 0.105) suggested NAC improved or preserved the hepatic circulation of the compromised liver. This NAC-induced improvement and (or) preservation of hepatic circulation was reflected in biliary and urinary excretion of acetaminophen and its metabolites by a general increase in elimination during the first 6 h (70.2 +/- 2.6 vs. 32.6 +/- 7.1%), and in the repletion of glutathione (GSH) in the liver by a return to control levels more quickly (3 vs. greater than 5 h) following depletion by NAPA. The metabolic consequences of the postabsorption antidotal effect of NAC in the compromised liver was a preferential excretion of sulphydryl-derived metabolites in the 1-4 h bile (GSH conjugate 11.30 +/- 1.25 vs. 7.25 +/- 0.39%) which was subsequently observed in the urine by preferential excretion of glutathione degradation products.

Isoniazid-induced hepatic steatosis in rabbits: an explanation for susceptibility and its antagonism by pyridoxine hydrochloride.

Steatosis was induced in rabbits by subacute administration of isoniazid (INH, 50 mg/kg po). Concomitant treatment with pyridoxine (vitamin B6, 25 mg/kg po) antagonized both development of the hepatic lesions and the elevation of plasma concentrations of lipids. Rabbit acetylating ability was sixfold that of male Wistar rats, a species susceptible to hepatic cell necrosis, whereas hepatic cytochrome P-450 and NADPH-cytochrome c reductase were significantly lower than that observed in control or phenobarbital-induced rats. Examination of the hepatic hydrolysis of the amide bonds of INH and acetylisoniazid (AcINH) indicated that the isonicotinoyl bond of AcINH was the bond most susceptible to amidase hydrolysis in both species; but rabbits possessed the greater amidase activity: 5- to 20-fold greater than control rats and 2- to 7-fold greater than the phenobarbital-induced rats. Consequently, INH-induced hepatic fatty degeneration in rabbits was attributed to increased hepatic exposure to INH-derived primary amine functional groups, and its antagonism by vitamin B6 was attributed to the deactivation of the primary amine by pyridoxal hydrazone formation.

Effect of salicylate on the fate of [14C]sulfinpyrazone in the rat.

The effect of acetylsalicylic acid (ASA, 100 mg/kg, po) on the fate of [14C]sulfinpyrazone (50 mg/kg, ip) was studied in male Wistar rats. ASA increased the rate of elimination of [14C]sulfinpyrazone-derived radioactivity from the blood during the first 5 h following drug administration. A tissue distribution study at 4 h showed that ASA increased the concentration of radioactivity in the gastrointestinal tract (GIT) and decreased it in the blood, heart, lung, and testis. ASA had little or no effect on the urinary and fecal excretion of sulfinpyrazone metabolites. Biliary elimination of radioactivity was increased from 54.2 +/- 4.7 to 69.6 +/- 1.2% of the dose in the 0- to 5-h period by salicylate (SAL), a major in vivo metabolite of ASA. Bile flow rate was also increased by SAL. An in vitro protein binding study showed that SAL displaced sulfinpyrazone from its binding sites. The results suggested that, in the rat, SAL enhanced the rate of sulfinpyrazone elimination by a combination of its choleretic action and displacement of bound sulfinpyrazone from plasma proteins.

Pathways of disposition of acetaminophen conjugates in the mouse.

After a single dose of [14C]acetaminophen (50 mg/kg) was administered orally to bile duct cannulated mice, 13.9% of the radioactivity was recovered in the bile while 41.2% was found in the urine in the first 3 h after administration. Analyses of biles revealed that the major biliary metabolite was acetaminophen glutathione (AG) conjugate which was derived from the hepatotoxic acetaminophen intermediate. Examination of urines showed that they contained mostly glucuronide and sulfate conjugates with no AG or its degradation products (cysteine and mercapturate). Analysis of urines collected from non-cannulated animals at 4 h showed that they contained glucuronide, sulfate, cysteine and mercapturate metabolites. Our results suggest that after formation in the liver, the majority of the glucuronide and sulfate conjugates were directly eliminated by the kidney. On the other hand, the pathway for the disposition of the glutathione conjugate was first into the bile, then reabsorption, and finally disposition into the urine as cysteine and mercapturate metabolites.

Isoniazid metabolism in the rabbit, and the effect of rifampin pretreatment.

The metabolism of 14C-isonicotinyl hydrazide (INH) (50 mg/kg, po) was studied in male New Zealand White rabbits and the effect on INH metabolism of pretreating the rabbits for 7 days with rifampin (100 mg/kg po per day) was also studied. The 14C-labelled metabolites were separated and quantitated by TLC and the unlabelled hydrazino metabolites by GLC. Absorption and elimination of INH was rapid since the peak blood 14C level was attained by 1 hr and the T 1/2 of elimination was 2.67 +/- 0.36 hr. By 12 hr 68.5 +/- 4.1% of the dose was recovered in the urine. The major metabolite excreted in the urine was isonicotinic acid (INA) which accounted for 40.3 +/- 3.5% of the dose followed by acetylisoniazid (AcINH) at 15.8 +/- 1.2%. The relatively high proportion of INA excreted by the rabbit compared to the rat and human is attributed to a high level of amidase in the rabbit, and is suggested as a possible explanation for the rabbit's sensitivity to INH-induced hepatotoxicity. Rifampin pretreatment produced only one significant change in the parameters studied and that was a reduction in AcINH excreted in urine. It is suggested that this effect may be due to rifampin increasing hepatic amidase activity.

N-acetylcysteine-induced inhibition of gastric emptying: a mechanism affording protection to mice from the hepatotoxicity of concomitantly administered acetaminophen.

Swiss Webster male mice, 22 +/- 3 g, killed 17-18 h following the concomitant oral administration of acetaminophen (350 mg/kg) and N-acetyl-cysteine (NAC, 100-500 mg/kg, treated) had statistically significant lower plasma transaminases (GOT and GPT) than control mice (acetaminophen + water). Possible mechanisms underlying this protective effect of NAC were examined. NAC (500 mg/kg) reduced [14C]acetaminophen-derived radioactivity in the blood and tissues but increased the percentage of the dose in the gastrointestinal tract. Depletion of hepatic sulphydryl compounds below 75% of the control value was prevented by NAC treatment, whereas urinary excretion of mercapturate and sulfate, metabolites derived from sulphydryls, were proportionally increased and excretion of unchanged drug was decreased by NAC. Absorption of acetaminophen from the small intestine was prevented by NAC and this was attributed to an inhibition in gastric emptying. Since all changes observed following NAC treatment could be attributed to inhibition of gastric emptying, it was considered the major mechanism responsible for affording in mice protection from acetaminophen-induced hepatocellular damage following concomitant oral administration.

Biliary and urinary excretion of [14C]warfarin in rabbits.

1. The biliary and urinary excretion of racemic [14C]warfarin (1 mg/kg) was studied in male rabbits. 2. In the first 6 h after dosing (i.v.), of the administered radioactivity was excreted warfarin and its hydroxylated metabolites. 3. Four days after oral dosing, about 90% of the administered radioactivity was recovered in the urine. The major urinary metabolites were identified as warfarin alcohols, unchanged warfarin, 6-, 7-, and 4'-hydroxywarfarin; their structures were unequivocally confirmed by g.l.c.-mass spectrometric analysis. 4. In the rabbit, the bile was only a minor excretory route for warfarin, and only polar and bulky conjugates of the drug were eliminated by this pathway.

Effect of a concomitant single dose of ethanol on the hepatotoxicity and metabolism of acetaminophen in mice.

A concomitant single dose of ethanol (1 g/kg) protected mice from hepatic injury induced by acetaminophen (250 mg/kg) as evidenced by the lowering of plasma transaminases. Pharmacokinetic studies with [14C]acetaminophen indicated that ethanol enhanced the initial blood concentrations of radiolabel and its rate of elimination. A tissue distribution study suggested that these effects were probably due to an ethanol-induced inhibition of the biliary clearance of acetaminophen from the blood. Examination of the urinary and biliary metabolites indicated that ethanol inhibited the excretion of the degradation products derived from the glutathione-deactivated hepatotoxic acetaminophen intermediate. The decrease in acetaminophen induced hepatotoxicity was therefore attributed to an inhibitory effect of ethanol on the biotransformation of acetaminophen to the toxic intermediate.

Fate of [14C]warfarin in guinea-pigs: effect of a concomitant single dose of salicylate.

When a single dose of sodium salicylate (177.8 mg kg-1, by mouth) was given with [14C] warfarin (1 mg kg-1, i.p.) to guinea-pigs, the salicylate depressed the blood concentrations of 14C for 6 h. At 1 h, salicylate increased the distribution of 14C in the liver and brain, but at 1 and 6 h it was decreased in the blood and kidney. A significant portion of the 14C was excreted into the bile, but was subject to enterohepatic circulation and then excreted by the kidney. There was an enhancement of the biliary elimination of 14C in the first 5 h after salicylate and a decrease in 14C concentration in blood; the proportion of warfarin to its metabolites excreted in the urine and bile was unchanged. Salicylate displaced serum protein bound [14C]warfarin in vitro. Salicylate increases the initial biliary elimination of warfarin by displacing some of that bound to plasma protein. This facilitated uptake of warfarin by the liver where it was metabolized. This effect of salicylate did not modify the hypoprothrombinaemia produced by warfarin.

Drug interactions with isoniazid metabolism in rats.

14C-Isoniazid (20 mg/kg po or iv) was administered alone or in combination with aspirin (100 mg/kg po), rifampin (30 mg/kg po), ethambutol (100 mg/kg po), or ethanol (3 g/kg po) to rats. In another experiment, phenobarbital sodium (40 mg/kg/day ip) was administered for 3 days prior to isoniazid. Aspirin and ethanol retarted the rate of isoniazid absorption from the GI tract. None of the drugs significantly altered the 14C-elimination rate from the blood over the first 4 hr. A tissue distribution study showed that changes in the blood levels produced by ethanol were reflected in the other tissues. When isoniazid was given intravenously, ethanol increased the amount of carbon-14 excreted in urine up to 24 hr after dosing; no other changes were observed in the total carbon-14 recovered in urine. Aspirin inhibited the conjugation of isonicotinic acid with glycine. Ethanol increased N-acetylisoniazid excretion and decreased isonicotinic acid excretion. None of the other treatments had more than a slight effect on isoniazid metabolism. Acute doses of isoniazid failed to produce any signs of hepatotoxicity, as judged by measurement of serum transaminase levels. The data do not suggest that any of the drugs studied are likely to potentiate the hepatotoxicity of isoniazid when administered acutely. Isoniazid metabolism in rats differed quantitatively from that reported for humans.

Analysis of warfarin in plasma by high-pressure liquid chromatography.

An improved high-pressure liquid chromatography method for the estimation of warfarin in plasma was developed. Plasma was acidified and extracted with ethylene dichloride spiked with methylated warfarin [3-(alpha-acetonylbenzyl)-4-methoxy-coumarin] as internal standard. The residue, redissolved in dioxane, was chromatographed on a reversed-phase column using a mobile phase of 40% dioxane in water (pH 4.2) on a high-pressure liquid chromatograph fitted with an UV absorbance detector. Recoveries from extraction, quantitated using tracer amounts of [14C]warfarin and methylated [14C]warfarin were 92.2 +/- 3.16% and 82.33 +/- 1.03%, respectively. The standard curve was linear between o.625 and 5.0 microng/ml. Detection was sensitive to approximately 0.5 microng/ml and specific without the inter ference of normal plasma constituents and warfarin metabolites.

Metabolism of [14C]paracetamol and its interactions with aspitin in hamsters.

1. The metabolism of [14C]paracetamol (150 mg/kg) and its interactions with aspirin (200 mg/kg) were studied in male hamsters. 2. Aspirin was found to slow the rate of paracetamol absorption from the gastro-intestinal tract, but did not affect the rate of elimination. 3. Metabolism studies showed that greater than 80% of the radioactivity was excreted in the urine in 24 h. Paper chromatography of the urine separated the radioactivity into five peaks, four of which were identified as paracetamol and its glucuronide, sulphate and mercapturate conjugates. 4. The other peak, comprising of less than 10% of the total radioactivity, was a mixture of two or more other metabolites. A major component was isolated and characterized as methyl 2-hydroxy-5-acetamidophenyl sulphone. 5. Aspirin inhibited the metabolism of paracetamol by the sulphate conjugation pathway.

High-pressure liquid chromatographic determination of acetaminophen in biological fluids.

A method for the rapid estimation of free acetaminophen in biological fluids is described. The assay involves either extraction and high-pressure liquid chromatographic analysis on a 10-mum particle-size silica gel column, using a mobile phase of 10% chloroform in tetrahydrofuran. The procedure was used to determine acetaminophen levels in urine from two healthy volunteers who ingested 650 mg of 14C-acetaminophen (20 muCi), and the accuracy of the method was compared with the carbon-14 determination. The limit of detectability for acetaminophen is 1 mug/ml.