Quantitative LC-MS determination of strychnine in urine after ingestion of a Strychnos nux-vomica preparation and its consequences in doping control.

A simple, fast and sensitive method for the quantitative determination of strychnine residues in urine has been developed and validated. The method consists of a liquid-liquid extraction step with ethyl acetate at pH 9.2, followed by LC-MS/MS in positive atmospheric pressure chemical ionization (APCI)-mode. The method is linear in the range of 1-100 ng/mL and allows for the determination of strychnine at sub-toxicological concentrations. The accuracy of the method ranged from 1.3% to 4.4%. The method was used to determine the excretion profile of strychnine after the ingestion of an over-the-counter herbal preparation of Strychnos nux-vomica. Each volunteer ingested a dose equivalent to 380 micro g of strychnine. This dose is lower than the prescription dose but results in the detection of strychnine for over 24-h post administration. Maximum detected urinary concentrations ranged from 22.6 to 176 ng/mL. The results of this study show that the use of this type of preparation by athletes can lead to a positive doping case.

GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine.

A procedure for the simultaneous confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine specimens by gas chromatography-mass spectrometry (GC-MS) is described. After the addition of nalorphine and naltrexone as the two internal standards, the urine is hydrolyzed overnight with beta-glucuronidase from E. coli. The urine is adjusted to pH 9 and extracted with 8% trifluoroethanol in methylene dichloride. After evaporating the organic, the residue is sequentially derivatized with 2% methoxyamine in pyridine, then with propionic anhydride. The ketone groups on hydrocodone, hydromorphone, oxycodone, oxymorphone, and naltrexone are converted to their respective methoximes. Available hydroxyl groups on the O3 and O6 positions are converted to propionic esters. After a brief purification step, the extracts are analyzed by GC-MS using full scan electron impact ionization. Nalorphine is used as the internal standard for codeine, morphine, and 6-acetylmorphine; naltrexone is used as the internal standard for the 6-keto-opioids. The method is linear to 2000 ng/mL for the 6-keto-opioids and to 5000 ng/mL for the others. The limit of quantitation is 25 ng/mL in hydrolyzed urine. Day-to-day precision at 300 and 1500 ng/mL ranged between 6 and 10.9%. The coefficients of variation for 6-acetylmorphine were 12% at both 30 and 150 ng/mL. A list of 38 other basic drugs or metabolites detected by this method is tabulated.

Automated solid-phase extraction of opiates from urine (I).

The extraction of morphine and codeine in urine by C18 bonded-phase columns was studied using a fully automated solid-phase extraction system. Extracts of urine were analysed by a high-performance liquid chromatographic method. Recovery of the opiates depends on the urine pH, and the choice of washing solvents and eluent. Studies were carried out on the optimisation of eluent volume by establishing a recovery-time profile for the elution process. Regenerated C18 bonded-phase columns could be re-used with negligible carry-over and insignificant recovery loss. An automated extraction procedure was developed for morphine and codeine in urine with absolute recoveries exceeding 80%.

Automated solid-phase extraction of opiates from urine (II). Establishing criteria for the detection of opiate abuse.

Automation in the laboratory was taken one step further with the introduction of automated solid-phase extraction of opiates from urine using C18 bonded-phase columns as an alternative to traditional liquid-liquid partitioning. A comparison of the automated procedure with an established liquid-liquid extraction procedure showed a linear correlation between the two over the concentration range studied. Criteria for the identification of opiate abusers were established to adapt this new methodology to large-scale screening of urine specimens.

Relation between renal and hepatic excretion of drugs: X. Excretion of nalorphine in young and adult rats pretreated with hormones or xenobiotics.

Different processes are involved in renal and hepatic excretion of organic anions and cations. In contrast to our knowledge of anion excretion, information about cation transport in kidney and liver is relatively scarce. In this study, the elimination of nalorphine was investigated to characterize the relation between renal and hepatic excretion of organic cations. Nalorphine is excreted effectively both via kidney and liver. However, its hepatic excretion dominates in adult rats. In young, 20-day-old animals biliary nalorphine elimination is immature and the excreted amounts are significantly lower. Renal excretion of nalorphine is quite similar in rats of both ages. After bile duct ligation renal excretion of nalorphine increases significantly in adult rats whereas it remains unchanged in young ones. Remarkably, after bilateral nephrectomy hepatic elimination of nalorphine is even diminished in both age groups. In further experiments renal excretion of nalorphine could be stimulated in adult rats after repeated administration of trometamol, triiodothyronine, or dexamethasone; these treatments had no consequences on biliary secretion of nalorphine.

Screening and confirmation of opiates by thin-layer chromatography.

A thin-layer chromatography (TLC) procedure for the screening and confirmation of urinary codeine and morphine has been developed. Urine samples are hydrolyzed to liberate free codeine and morphine, extracted into an organic solvent, and concentrated. For screening, the extraction residues are spotted on TLC plates which are developed in chloroform:ethyl acetate:methanol:propylamine (35:45:5:5). For confirmation, the extraction residues are acetylated and then spotted on TLC plates which are developed in hexane:chloroform:diethylamine (50:30:7). The spots for the two drugs are visualized with acidified iodoplatinate. Codeine and morphine are well separated from one another and from normal urinary substances on both plates and can be detected at a concentration of 0.4 micrograms/mL.