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.

Renal accumulation of salicylate and phenacetin: possible mechanisms in the nephropathy of analgesic abuse.

Since either aspirin or phenacetin might be causative in the nephropathy of analgesic abuse, studies were designed to examine the renal accumulation and distribution of the major metabolic products of these compounds, salicylate and N-acetyl-p-aminophenol (APAP) respectively, in dogs. Nineteen hydropenic animals were studied, of which seven were given phenacetin, nine received acetyl salicylic acid, two were given both aspirin and phenacetin, and one received APAP directly. Two of three hydrated animals were given phenacetin and one was given aspirin. During peak blood levels of salicylate and (or) APAP, the kidneys were rapidly removed, frozen, sliced from cortex to papillary tip, and analyzed for water, urea, APAP, and salicylate. No renal medullary gradient for salicylate was demonstrable during both hydropenic and hydrated states. In contrast, both free and conjugated APAP concentrations rose sharply in the inner medulla during hydropenia, reaching a mean maximal value at the papillary tip exceeding 10 times the cortical concentration (P < 0.001), a distribution similar to that of urea. Salicylate had no effect on the APAP gradient, but hydration markedly reduced both the APAP and urea gradients in the medulla. The data indicate that APAP probably shares the same renal mechanisms of transport and accumulation as urea and acetamide, and that papillary necrosis from excessive phenacetin may be related to high papillary concentration of APAP.

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.

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.

Post-transplantation analgesic dependence in patients who formerly suffered from analgesic neophropathy.

Twenty-two patients with functioning grafts who originally developed renal failure due to analgesic nephropathy ("analgesic" group), and 84 patients with various other causes of renal failure ("non-analgesic" group) were studied over one year to assess the extent of analgesic use and abuse. On each occasion that one of these patients reported to hospital a urine sample was collected and analyzed for N-acetylparaminophenol (NAPA). Of the "analgesic" group of patients, 14% had consistently negative urine samples while 41% showed NAPA in more than half the urines collected. In the "non-analgesic" group 29% of patients had entirely negative urine tests, and in only 7% were more than half of the urines NAPA-positive. Patients in the "non-analgesic" group readily admitted taking anaglesics at frequencies compatible with the observed number of positive tests. This contrasted with the "analgesic" group in which 14 of 19 patients with NAPA-positive urines denied analgesic intake. This is considered to be a guilt manifestation in patients who have developed a psychological dependence on analgesics and have recidivated with full knowledge of the possible harm these drugs may inflict on their grafts.

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.

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.

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.

[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.