Immunoassay and GC-MS procedures for the analysis of drugs of abuse in meconium.

The analysis of meconium specimens for metabolites of substances of abuse is a relatively accurate method for the detection of fetal exposure to drugs. Most of the methods reported in the literature before the early 1990s relied on radioimmunoassays. The purpose of this study was to develop and validate methods for meconium sample preparation for the screening and gas chromatography-mass spectrometry (GC-MS) confirmation of meconium extracts for cannabinoids, cocaine, opiates, amphetamines, and phencyclidine. EMIT and TDx immunoassays were evaluated as screening methods. The sample preparation method developed for screening included extraction and purification prior to analysis. Cutoff levels were administratively set at 20 ng/g for 11-nor-delta9-THC-9-COOH (THCCOOH) and phencyclidine and at 200 ng/g for benzoylecgonine, morphine, and amphetamines, although lower levels could be detected in meconium using the EMIT-ETS system. Ninety-five meconium specimens were subjected to the screening procedure with GC-MS confirmation of presumptive positives. In addition, 30 (40 for cocaine) meconium specimens were subjected to GC-MS analysis for all analytes regardless of the screening results to determine the false-negative rate, if any, of the immunoassay. Although there were no false negatives detected, the GC-MS confirmation rate for the immunoassay-positive specimens was generally low, ranging from 0% for amphetamines to 75% for opiates. The lowest rate of confirmed positives was found with the cannabinoids, suggesting that tetrahydrocannabinol (THC) metabolites other than free 11-nor-9-carboxy-delta9-THC may be major contributors to the immunoassay response in meconium.

Hexadeutero-11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid: a superior internal standard for the GC/MS analysis of delta 9-THC acid metabolite in biological specimens.

GC/MS analysis of biological specimens is believed to be the most forensically accepted method for confirming the presence of abused drugs. 11-Nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC-COOH) is the major metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) for which testing (including GC/MS) is directed as an indication of marijuana use. The currently available internal standard for delta 9-THC-COOH is d3-delta 9-THC-COOH, which has the deuterium atoms located on the side chain. In addition to the high cost of this compound, it suffers from a limited dynamic range of analysis, especially when the methyl derivative is used. This is because of a contribution to one of the internal standard ions (m/z 316) from a fragmentation of the natural drug which involves loss of the side chain. The new internal standard, d6-11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (d6-delta 9-THC-COOH), avoids these disadvantages. The six deuterium atoms are located on the two methyl groups of Carbon 6 in the dibenzopyran structure. The dynamic range of analysis with the new internal standard was tested between 6.25 to 1,000 ng/mL with a correlation coefficient of 0.998. Analysis of several urine specimens for delta 9-THC metabolite using both d3- and d6-internal standards showed a correlation coefficient of 0.9987.

Rectal bioavailability of delta-9-tetrahydrocannabinol from various esters.

The bioavailability of delta-9-tetrahydrocannabinol (delta 9-THC) from suppository formulations containing several polar esters was studied. The esters tested were the hemisuccinate, N-formyl alaninate, N-methyl carbamate, and methoxy acetate. These esters were administered to monkeys in both lipophilic and hydrophilic suppository bases, namely, Witepsol H15 and polyethylene glycol, respectively. Each suppository contained a dose equivalent to 10 mg delta 9-THC. Blood samples were analyzed for both delta 9-THC and its carboxylic acid metabolite (ll-nor-delta 9-THC-9-COOH) using gas chromatography/mass spectrometry. The data showed that, with the exception of the hemisuccinate, no delta 9-THC or its metabolite was detected in the blood samples using the Witepsol H15. Using polyethylene glycol, low levels of delta 9-THC and its metabolite were detected in blood for all esters tested. The levels, however, were lower than those observed with delta 9-THC hemisuccinate using Witepsol H15. Subsequent studies in the conscious dog using the hemisuccinate in Witepsol H15 showed 67% bioavailability of delta 9-THC with a linear response in the dose range equivalent to 5-20 mg of delta 9-THC. No significant bioavailability differences were found when delta 9-THC hemisuccinate ester was administered in various lipophilic bases (Hydrokote 25, Kaomel, Suppocire AIML, and Witepsol H15).

Rectal bioavailability of delta-9-tetrahydrocannabinol from the hemisuccinate ester in monkeys.

Oral administration of delta-9-tetrahydrocannabinal (delta 9-THC) was shown to result in low and erratic bioavailability, while the drug showed no bioavailability from various suppository formulations. delta 9-THC-Hemisuccinate was formulated as a prodrug for delta 9-THC in suppositories using Witepsol H15 base. The bioavailability of delta 9-THC from this formulation was evaluated in monkeys. The plasma levels of delta 9-THC and its metabolite 11-nor-delta 9-THC-9-COOH were determined using GC/MS analysis. The calculated bioavailability of delta 9-THC from this formulation was found to be 13.5%. Non-compartmental analysis of the plasma concentration data using statistical moments showed the mean residence time (MRT) for delta 9-THC in the body to be 3 h following iv administration of delta 9-THC or its hemisuccinate ester (3.4 and 2.7 h, respectively), as compared with 5.8 h following rectal administration of the delta 9-THC hemisuccinate. The observed rectal bioavailability of delta 9-THC from suppositories containing the hemisuccinate ester as a prodrug is of significant importance in developing an alternative approach to oral administration of the drug.

Poppy seed ingestion and opiates urinalysis: a closer look.

Review of scientific literature shows that ingestion of poppy seed containing products can result in a positive urinalysis test for opiates. In many cases the amount of seeds ingested is unrealistically high or is not specified. This study is designed to correlate the amount of seeds ingested with the urinary concentration of total morphine as a function of time. Two males and two females were involved in all four protocols, which were separated by at least one week. Subjects ingested one, two, or three poppy seed rolls, each containing 2 g of Australian seeds (108 micrograms morphine/g seed) in three protocols. In the fourth protocol subjects ingested two rolls per day for four consecutive days. Urine specimens were collected for 48 h after ingestion, analyzed by RIA, EMIT, and TDx, and selected samples were confirmed by GC/MS. The data show that the highest concentrations of total morphine in urine were found 3-8 h after ingestion or in the first-void samples. Of the 264 samples collected, there were only 16 specimens that exceeded 300 ng/mL by any of the methods used for analysis with only three samples exceeding 400 ng/mL by GC/MS (406, 611, and 954 ng/mL). In all cases, the total opiates level was less than 150 ng/mL 24 h after ingestion. Following these studies, one of the subjects ingested a poppy seed cake containing 15 g seed obtained from a bakery which analyzed for 169 micrograms morphine/g seed. Urine specimens were collected over 48 h, and all specimens were analyzed by GC/MS.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas chromatographic/mass spectrometric analysis of morphine and codeine in human urine of poppy seed eaters.

In this study, poppy seeds were examined for a natural constituent that might serve as a maker for the seeds' ingestion as opposed to opiate abuse. Thebaine was selected as possible marker, since it was found to be a component of all poppy seeds examined and was not a natural component of different heroin samples. During the course of this investigation, a new extraction and cleanup procedure was developed for the gas chromatographic/nitrogen phosphorus detection (GC/NPD) and gas chromatographic/mass spectrometric (GC/MS) analysis of morphine and codeine in urine. A linear response, over a concentration range of 25 to 600 ng/mL, was obtained for codeine and morphine (r = 0.9982 and 0.9947, respectively). The minimum detectable level (LOD) and limit of quantitation (LOQ) for morphine were 10 and 30 ng/mL, respectively; whereas LOD and LOQ for codeine were 2 and 8 ng/mL, respectively. The coefficients of variance (CV, n = 6) for morphine and codeine analyses at the 100-ng/mL level were 13.3 and 4.6%, respectively. This procedure was used for the analysis of urine samples from five poppy seed eaters who each ingested 200 g of poppy seed cake. Results indicated that significant amounts of morphine and codeine are excreted in urine and that in all subjects, at least at one point in time, the apparent morphine concentration as determined by radioimmunoassay (RIA) analysis exceeded the cutoff value (300 ng/mL) established for screening. Thebaine was not detected in urine specimens collected following poppy seeds ingestion and thus could not be used as a marker.