Determination of cocaine adulterants in human urine by dispersive liquid-liquid microextraction and high-performance liquid chromatography.

This study aimed to determine simultaneously five major street cocaine adulterants (caffeine, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine by dispersive liquid-liquid microextraction (DLLME) and high-performance liquid chromatography. The chromatographic separation was obtained in gradient elution mode using methanol:water plus trifluoroacetic acid 0.15% (v/v) (pH = 1.9) at 1 mL min-1 as mobile phase, at 25 °C, detection at 235 nm, and analysis time of 20 min. The effect of major DLLME operating parameters on extraction efficiency was explored using the multifactorial experimental design approach. The optimum extraction condition was set as 4 mL human urine sample alkalized with 0.5 M sodium phosphate buffer (pH 12), NaCl (15%, m/v), 300 µL acetonitrile (dispersive solvent), and 800 µL chloroform (extraction solvent). Linear response (r2 ≥ 0.99) was obtained in the range of 180-1500 ng mL-1 with suitable selectivity, quantification limit (180 ng mL-1), mean recoveries (33.43-76.63%), and showing relative standard deviation and error (within and between-day assays) ≤15%. The analytes were stable after a freeze-thaw cycle and a short-term room temperature stability test. This method was successfully applied in real samples of cocaine users, suggesting that our study may contribute to the appropriate treatment of cocaine dependence or with the cases of cocaine acute intoxication.

GC/MS confirmation of barbiturates in blood and urine.

A gas chromatography-mass spectrometric method is described for the quantitative measurement of 6 commonly used barbiturates in blood and urine specimens. The targeted barbiturates are butalbital, amobarbital, pentobarbital, secobarbital, mephobarbital and phenobarbital. They are recovered along with the internal standard, tolybarb, from blood and urine using liquid extraction then alkalated to form the N-ethyl derivatives. The ethylated barbiturates have symmetrical peaks which are well separated from each other on a non-polar methylsilicone capillary column. The derivatives on a non-polar methylsilicone capillary column. The derivatives facilitate quantitations between 50 and 10,000 ng/mL. The day-to-day CVs for all 6 barbiturates were between 4 and 9% at 200 and 5000 ng/mL. The method has been extended for identifying other acidic drugs and drug metabolites. They are mainly non-steroidal anti-inflammatory drugs, diuretics, and anticonvulsants. An additional 83 compounds can be qualitatively identified.

NMR spectroscopic studies on the metabolism and futile deacetylation of phenacetin in the rat.

1. 1H-NMR spectroscopy of urine was used to determine the % deacetylation and re-acetylation of 2H-labelled (in the acetyl) phenacetin metabolites in the rat. 2. Male Sprague-Dawley rats were each dosed with either phenacetin or phenacetin-C2H3 at 50 mg kg-1. The total urinary recoveries for phenacetin and phenacetin-C2H3 were 47.6 +/- 16.7 and 50.1 +/- 16.2% respectively (not significantly different, p > 0.05). Paracetamol sulphate and glucuronide are the major urinary metabolites of both protio and deuteriophenacetin. 3. The futile deacetylation given by the urinary recovery of protio-acetyl metabolites of phenacetin-C2H3 was 29.6 +/- 0.9% for paracetamol sulphate and 36.6 +/- 3.1% for paracetamol glucuronide. These observations demonstrate a high level of futile deacetylation in the paracetamol conjugates formed by metabolism of phenacetin-C2H3 and this may indicate a high metabolic flux through the nephrotoxic intermediate 4-aminophenol. 4. The level of futile deacetylation for phenacetin was significantly higher than that found previously in studies of labelled paracetamol in rat or man, and may be important in understanding the higher nephrotoxicity of phenacetin as compared with paracetamol.

Studies on 13C-phenacetin metabolism. II. A combination of breath test and urine test of in vivo metabolites in the diagnosis of liver disease.

We determined the optimum 13C-labeling position in phenacetin for use in a breath test to diagnose liver disease based on infrared spectroscopy detection of 13CO2 in exhaled air. ([1-13C]Ethoxy)phenacetin gave the best result. This compound was also employed in a urine test using 13C-NMR spectroscopy. In the urine test, healthy subjects gave a higher signal of phenacetin than of its metabolite, phenetidine, whereas in patients with liver disease the situation was the reverse. The combination of the breath and urine tests may be a valuable new tool for the diagnosis of liver disease.

Influence of phenacetin and its metabolites on renal function.

To investigate the pathogenesis of phenacetin-induced nephropathy, the influence of aspirin and caffeine on phenacetin metabolism was studied. Eight healthy male volunteers participated in the study after giving written consent. They were randomly divided into 2 groups. Phenacetin was given to one group, and phenacetin, aspirin and caffeine were given to the other group based on a cross-over design. Blood and urine were collected over a period of 24 hours. The urinary excretion and plasma concentrations of unchanged phenacetin, acetaminophen, acetaminophen glucuronide and acetaminophen sulphate were measured by high performance liquid chromatography. The proportions of urinary excretion of these substances were not significantly different in the two groups. The pharmacokinetic parameters of these substances were also fundamentally identical. It may be concluded that aspirin and caffeine do not alter the phenacetin metabolism. However, other minor metabolites such as p-phenetidine must be closely investigated before we can draw any final conclusions.

The metabolism of N-hydroxyphenacetin in vitro and in vivo.

N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2.1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1.8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0.4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP) (0.05%) were found. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.

Formation of reactive metabolites of phenacetin in humans and rats.

The metabolism of phenacetin to reactive intermediates in humans was estimated from the excretion of thio adducts in urine. N-Hydroxyphenacetin, a precursor of reactive metabolites, was also quantified. Following an oral dose of phenacetin (10 mg/kg) to humans, these metabolites in 24 h urine were: paracetamol-3-cysteine, 4.4% dose; paracetamol-3-mercapturate, 3.9%; 3-thiomethylparacetamol, 0.4%; N-hydroxyphenacetin, 0.5%. Rats showed a considerable increase in N-hydroxyphenacetin excretion after chronic dosing with phenacetin at high dosage (500 mg/kg) for one month. chronic dosing with a low dose (50 mg/kg) did not increase N-hydroxyphenacetin excretion, but a marked increase occurred on concomitant administration of aspirin and caffeine.

Availability studies on acetylsalicylic acid, salicylamide and phenacetin at different pH values.

The optimum partitioning rate of acetylsalicylic acid has been attained at pH = 4 and minimum partitioning rate was found to be at pH = 8. The maximum partitioning rate of salicylamide was observed at pH = 5 and the smallest one was found at pH = 6 or 8. At pH = 3 a maximum amount of phenacetin was found in the aqueous phase, while at pH = 6 a maximum amount was found in the octanolic layer. The maximum partitioning rate was found at pH = 6 and lowest one was observed at pH = 3. The gastrointestinal absorption of acetylsalicylic acid, salicylamide and phenacetin was significantly increased, as reflected by the urinary excretion data in presence of solid buffer components at pH values of 4,5 and 6 respectively.

Mass spectrometric determination of N-hydroxyphenacetin in urine using multiple metastable peak monitoring following thin-layer chromatography.

This work describes a method for the quantitative determination of the labile, toxic N-hydroxy metabolite of phenacetin in urine. A thin-layer chromatography step was used for the preliminary purification of extracts, and the specificity of the assay was based on the monitoring of specific metastable decompositions in a forward geometry double-focussing mass spectrometer, in a manner analogous to conventional tandem mass spectrometry. This precluded the need for a gas chromatographic separation, thus minimizing thermal decomposition which can occur with these compounds, as well as enabling very rapid analyses.

Species-specific activation of phenacetin into bacterial mutagens by hamster liver enzymes and identification of N-hydroxyphenacetin O-glucuronide as a promutagen in the urine.

Phenacetin was mutagenic in Salmonella typhimurium TA100 in plate assays when liver fractions from Aroclor-treated hamsters, but not rats, were used. Its known or putative metabolites were synthesized; of these, N-hydroxyphenacetin and N-acetoxyphenacetin were found to be mutagenic in liquid and plate assays, both requiring activation by liver fractions from Aroclor-treated hamsters. 2-Hydroxyphenacetin and 2-acetoxyphenacetin were nonmutagenic. N-Hydroxyphenetidine (the deacetylated metabolite of phenacetin) and p-nitrosophenetole were the only products that were found to be mutagenic per se when assayed under N2 in either Salmonella TA100 and TA100 NR (nitroreductase-deficient) strains. Phenacetin was administered to male BDVI rats and Syrian golden hamsters, and its urinary metabolites were deconjugated with beta-glucuronidase:arylsulfatase. After reactivation by hamsters liver fractions, mutagenicity was demonstrated in S. typhimurium TA100 with urine from phenacetin-treated hamsters, but not with that from rats. After treatment with deconjugating enzymes, N-hydroxyphenacetin was isolated from hamster urine by high-performance liquid chromatography and identified by mass spectral analysis. The data support the conclusions that (a) N-hydroxyphenacetin is a proximate mutagenic metabolite of phenacetin which, after N-deacylation, is responsible for the mutagenicity observed in vitro and in the urine of hamsters and (b) the higher yield of N-hydroxyphenacetin that is formed in the liver of hamsters as compared to rats explains the pronounced species-specific activation of phenacetin into bacterial mutagens.

N-hydroxyphenacetin, a new urinary metabolite of phenacetin in the rat.

N-Hydroxyphenacetin has been found in the urine of rats dosed with phenacetin, extending previous reports that phenacetin is N-hydroxylated by liver microsomes in vitro. After an oral dose of phenacetin (500 mg/kg) urine was collected for 24 hr, conjugates hydrolyzed with extract of Helix pomatia, and the metabolites extracted with dichloromethane and treated with diazomethane. Methylation of N-hydroxyphenacetin produced a stable derivative, N-methoxyphenacetin, which was separated from most other metabolites by thin layer chromatography. Identification of N-methoxyphenacetin was by combined gas chromatography-mass spectrometry and comparison with the synthetic reference compound. Quantification by gas chromatography with flame-ionization detection showed that 0.023% of the dose phenacetin was recovered from urine as N-hydroxyphenacetin. It is probable that this value considerably underestimates the extent of phenacetin N-hydroxylation in vivo, inasmuch as N-hydroxyphenacetin is known to be rapidly degraded in biological systems.

[Finding of analgesic components and their metabolites in doping control]. / Nálex soucástí analgetik a jejich metabolitu pri dopingové kontrole.

In doping controls it is important to exclude essential substances that are not in the list of forbidden drugs. Similarly it is important as well for these drugs to be proved in analgesic mixture abuse. An example being the proof of amidopyrine and its metabolites, further on p-phenetidine as a metabolie of phenacetin in alkaline urine extracts. Results are given in using thin-layer chromatography, gas chromatography and the identification by means of gas chromatography/mass spectrometry.

4-Acetaminophenoxyacetic acid, a new urinary metabolite of phenacetin.

It is shown that 4-acetaminophenoxyacetic acid (APOA) is an urinary metabolite of phenacetin. APOA was isolated by means of silica gel TLC in various solvent systems from the urine of rats, dogs, and humans, collected 24 h after p.o. treatment with phenacetin (rats and dogs: 200 mg/kg; humans: three single doses of 0.5 g). Expressed as a percentage of the dose, APOA was detected at levels of 1% in rats, 0.13% in dogs and 0.04% in humans. 4-Acetaminophenoxyacetic acid was identified as its methylester--synthetized in the reaction of APOA and diazomethene--by thin layer chromatography, UV absorbance, melting point, and mass spectroscopy.

Phenacetin: renal tubular transport and intrarenal distribution in the dog.

In a total of 22 studies in the anesthetized dog, the renal clearnace of phenacetin was measured over a range of plasma concentrations and at different urinary pH and rates of urine flow. Phenacetin is reabsorbed by passive diffusion. The urine/plasma ratio is essentially unity under all conditions. As measured by ultraviolet-spectrophotometric and colorometric methods and by high-pressure liquid chromatography, a labile metabolite appears in urine and renal papilla which upon hydrolysis gives rise to falsely high values for phenacetin. Renal cortical and papillary concentrations were determined in six dogs during hydropenia or diuresis. All tissue/plasma concentration ratios for phenacetin were essentially unity. The results indicate that the papillary localization of the earliest lesions of analgesic nephropathy cannot be attributed to high phenacetin concentrations within the papilla. This is in contrast to acetaminophen, the major metabolite of phenacetin.