Comparison of drugs of abuse detection in meconium by EMIT II and ELISA.

The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme multiplied immunoassay technique (EMIT((R) )II) and enzyme-linked immunosorbent assay (ELISA) methods were compared. The sample preparation for the ELISA screen was a simple buffer extraction versus a lengthy and more laborious sample preparation procedure for the EMIT II screen. The ELISA method was automated using a TECAN Genesis. The EMIT II analysis was automated with an Olympus AU400e. The opioid screen was calibrated with hydromorphone and the benzodiazepine screen was calibrated with clonazepam to maximize detection for these analytes. Previously validated gas chromatography-mass spectrometry (GC-MS), two-dimensional GC-MS, or liquid chromatography-tandem MS methods were used for confirmation. Results from the two techniques compared well. Agreement of the ELISA assay was greater than 90% when compared to EMIT II for all drug classes except barbiturates and benzodiazepines. ELISA appears to be more sensitive than EMIT II for the detection of amphetamines, methadone, propoxyphene, and cocaine. ELISA compared well to EMIT II for cannabinoids, opioids, and PCP. Specificity of the ELISA assay was slightly better for PCP and opioids. EMIT II appears to be more sensitive for the detection of barbiturates and benzodiazepines. The ELISA method reduced turnaround time by 50% compared to the EMIT II method.

Retrospective diagnosis of an adverse drug reaction in a breastfed neonate: liquid chromatography-tandem mass spectrometry quantification of dextropropoxyphene and norpropoxyphene in newborn and maternal hair.

Dextropropoxyphene (DP) and norpropoxyphene (NP) are commonly used in the treatment of postpartum pain. The drug is widely prescribed in Europe and Canada and has been recently approved for use in the U.S. Its safety during breastfeeding, however, has not been fully established. Very few reports on its effects on neonates have been published. We report here the case of a mother treated with DP (6 capsules a day for 10 days) while she was breastfeeding. On day 7, her baby was lethargic and had difficulties with breastfeeding, which led to early weaning. The correlation between side effects observed in the infant and DP was made retrospectively by measuring DP and NP hair concentrations in the mother-infant pair with liquid chromatography-tandem mass spectrometry. Breastfeeding mothers taking DP expose their infants to high doses of DP and NP. In agreement with previously published reports, these data indicate that acetaminophen and nonsteroidal antiinflammatories are preferable for analgesia during breastfeeding. Breastfeeding should be encouraged under most circumstances, and if the mother takes any treatment for pain, a commonly prescribed drug with pharmacologic data available must be used.

Determination of propoxyphene in oral fluid.

The determination of propoxyphene in oral fluid using solid-phase extraction and gas chromatography-mass spectrometry is described for the first time. The method employs collection of oral fluid with the Quantisal device, immunoassay screening of the specimen, confirmation of the positive screened samples after extraction using cation exchange/hydrophobic solid-phase extraction columns, optimized derivative formation, and gas chromatography-mass spectrometry in electron impact mode. Validated parameters including selectivity, linearity, accuracy, intra- and interday precision, extraction efficiency, and limit of quantitation were all within acceptable limits. The method was applied to authentic specimens taken from an individual prescribed propoxyphene following surgery.

Determination of propoxyphene and norpropoxyphene by chemical ionization mass fragmentography.

The kinetics of propoxyphene and its primary metabolite, norpropoxyphene, have been simultaneously re-evaluated in man by using gas-liquid chromatography/mass spectrometry, deuterium-labeled mass internal standards, and multiple ion monitoring. Plasma concentrations in 4 volunteers determined for as long as 240 hr after single oral doses of 2 propoxyphene salts indicate the overall half-life of propoxyphene to be 11.8 hr and of norpropoxyphene to be 36.6 hr.

Liquid chromatographic determination of propoxyphene and norpropoxyphene in plasma and breast milk.

A sensitive and specific high-performance liquid chromatographic (HPLC) procedure was developed for determination of propoxyphene and norpropoxyphene in plasma and breast milk. The compounds were isolated from the biological specimen by extraction, the organic phase was evaporated to dryness, and the residue was redissolved in mobile phase [acetonitrile: 0.002 M H2SO4 (1:1)]. The resultant solution was then injected into an HPLC system utilizing a C18 reversed-phase analytical column and a variable-wavelength detector set at 205 nm. Under these conditions the method has a sensitivity of 20 ng/mL using 1 mL of plasma or milk. The within-run coefficient of variation for both compounds varied between 6.2 and 8.9% within the concentration range tested. Applicability of the method was demonstrated in a nursing mother who received multiple oral doses of propoxyphene.

Screening for drugs of abuse (II): Cannabinoids, lysergic acid diethylamide, buprenorphine, methadone, barbiturates, benzodiazepines and other drugs.

Requirements for the provision of an efficient and reliable service for drugs of abuse screening in urine have been summarized in Part I of this review. The requirements included rapid turn-around times, good communications between requesting clinicians and the laboratory, and participation in quality assessment schemes. In addition, the need for checking/confirmation of positive results obtained for preliminary screening methods was stressed. This aspect of the service has assumed even greater importance with widespread use of dip-stick technology and the increasing number of reasons for which drug screening is performed. Many of these additional uses of drug screening have possible serious legal implications, for example, screening school pupils, professional footballers, parents involved in child custody cases, persons applying for renewal of a driving licence after disqualification for a drug-related offence, doctors seeking re-registration after removal for drug abuse, and checking for compliance with terms of probation orders; as well as pre-employment screening and work-place testing. In many cases these requests will be received from a general practitioner or drug clinic with no indication of the reason for which testing has been requested. This also raises the serious problems of a chain of custody, provision of two samples, stability of samples, and secure and lengthy storage of samples in the laboratory-samples may be requested by legal authorities several months after the initial testing. The need for confirmation of positive results is now widely accepted but it may be equally important to confirm unexpected negative results. Failure to detect the presence of maintenance drugs may lead to the patient being discharged from a drug treatment clinic and, if attendance at the clinic is one of the terms of continued employment, to dismissal. It seems likely that increasing abuse of drugs and the efforts of regulatory authorities to control this, will lead to the manufacture of more designer drugs. Production of substituted phenethylamines was facilitated by the drug makers' cook book, 'PIHKAL' (Phenethylamines I Have Known And Loved) by Dr Alexander Shulgin and Ann Shulgin, and production of substituted tryptamines is promised in their next book, TIHKAL. Looking to the future, laboratories will need to ensure that they can detect and quantitate an ever-increasing number of drugs and related substances. The question of confidence in results of drugs of abuse testing raised in 1993 by Watson has assumed even greater importance as a result of attention focused on the OJ Simpson trial in Los Angeles. Toxicological investigations are likely to be challenged more frequently in the future. Even if analyses have been performed by GC-MS, there is a need to establish the level of match between the spectrum of the unknown substance and a library spectrum which is considered acceptable for legal purposes. It will also be essential to ensure that computer libraries contain spectra for all substances likely to be encountered in drugs of abuse screening.

Catalytic tritiation of drugs and analysis of the tritium distribution by 3H n.m.r. spectroscopy.

Propranolol, phenobartitone, diphenylhydantoin, amphetamine, imipramine and propoxyphene have been tritiated using tritiated water and platinum catalyst (from the dioxide and sodium borohydride), and the pattern of labelling has been ascertained by 3H n.m.r. spectroscopy. The results show that this exchange procedure can lead to satisfactory incorporation of tritium at 'stable' aromatic positions. For imipramine where incoroporation of tritium was low, an alternative acid-catalysed procedure was satisfactory.

Quantification of dextropropoxyphene and its metabolite by HPLC in hair of overdose cases.

Hair samples taken from 11 persons suspected to have died from an overdose of legal or illicit drugs were analysed. These hair samples were selected, because classical post-mortem toxicological investigations in blood revealed the presence of dextropropoxyphene (PPX) and its major metabolite norpropoxyphene (NPPX). For the hair analysis, hair strands were cut into segments of about 3 cm, washed with water and acetone, dried and pulverised. An aliquot of about 40 mg of those segments was incubated with acetate buffer pH 4.0 and beta-glucuronidase/arylsulfatase. After liquid-liquid extraction with hexane-3-methylbutanol (99:1), drugs were identified and measured by HPLC using piritramide as an internal standard. Preliminary assays showed that the limit of detection for PPX is below 1.0 ng/mg hair, the limit of detection for the metabolite NPPX being significantly higher (1.5 ng/mg). GC/MS, usually the method of choice for this kind of determination, was not chosen, because of the thermolability of PPX and its unspecific mass spectrum. From the hair of 11 persons, 24 segments were prepared and processed. Our results show that ten persons were found positive for PPX; moreover, when regarding the 24 segments, only three were found negative. PPX and NPPX concentrations were detected at maximal concentrations of 26.4 and 71.0 ng/mg hair respectively. Considering the ratio of PPX to NPPX for each person, we found more PPX than NPPX for three persons, whereas for seven persons we found more of the metabolite than its parent drug.

Toxicological findings in a multi-drug death involving propoxyphene, caffeine, phenacetin, acetaminophen and salicylate.

The toxicological findings of a multi-drug related fatal poisoning are described here. A 35-year-old Caucasian male found dead on the kitchen floor was a known user of abused drugs and had been taking aspirin alone or in combination with phenacetin and caffeine for the relief of joint pains. The gross examination of the organs at autopsy revealed slight grooving of the uncus and various stages of necrosis in the renal papillae. Histological examination confirmed the gross appearance of pulmonary congestion and edema, cerebral edema and interstitial nephritis of the tubules. Toxicological evaluation of the blood and urine samples disclosed the presence of propoxyphene (51 and 250 mg/l), salicylate (185 and 2750 mg/l), caffeine (16 and 37 mg/l), and phenacetin (9.6 and 20 mg/l). Furthermore, acetaminophen also was present in the plasma (54 mg/l) and urine. A gas liquid chromatographic method for simultaneous analysis of phenacetin and caffeine utilizing a nitrogen phosphorus detector was proposed.

Evaluating suspected co-proxamol overdose.

In three instances of suicidal poisoning by co-proxamol (paracetamol and dextropropoxyphene) blood samples were obtained from 11 sites together with eight tissue samples, bile, urine, gastric contents and duodenal contents. Site-dependent differences in blood propoxyphene concentration varied between the three cases but concentrations were consistently lowest in peripheral blood and highest in central sites: 3.9-5.5 (pulmonary vein) mg/l; 4.6-25 (pulmonary vein) mg/l; 3.2-40 (aorta) mg/l. There was a less than twofold variation in corresponding blood paracetamol concentrations. Reference data on fatal propoxyphene blood concentrations do not specify the blood sampling site and can be misleading. The intra-individual variability of propoxyphene concentrations in blood in these three cases underscores this problem. Tissue concentrations of propoxyphene showed considerable inter-individual variability in degree and pattern. Tissue concentrations of paracetamol showed a less than twofold intra-individual variation. Body drug loads were calculated by two methods: from organ weights and tissue concentrations; from published volume of distribution data (Vd). For paracetamol the body drug load is underestimated by the organ weight calculation but the Vd calculation approximates the suspected dose based on anamnestic information. For propoxyphene the body drug load is seriously underestimated by the organ weight calculation and overestimated up to 2.5 times by the Vd calculation. Since the two drugs have a fixed ratio in co-proxamol then the dose of propoxyphene (the effective lethal agent) can be inferred from the paracetamol dose calculated by Vd. This approach may be applicable to cases of overdose with other compounded drug preparations.

Detection of methadone and propoxyphene in stored tissue.

The effect of time and storage on the detection of methadone and propoxyphene in tissue was investigated. Specimens obtained at autopsy were stored: 1) at -12 degrees C, and 2) in 10% formalin at 4 degrees C. Aliquots of these tissues were analyzed following autopsy and at intervals thereafter. Other samples were analyzed at intervals without an initial perimortem study. The aliquots were assessed by gas chromatography and Enzyme Multiplied Immuno-assay Technique (EMIT), Methadone and propoxyphene were detected in preserved tissue 19 and 24 months after death. The primary value of the EMIT technique was to confirm the drug identified and quantitated by GC.

Abbott propoxyphene assay: evaluation and comparison of TDx FPIA and GC/MS methods.

This study evaluated the capability of the Abbott TDx assay to test for propoxyphene in urine and various biological samples, including tissues obtained from three fatal overdoses, by comparison to gas chromatography/mass spectrometry (GC/MS). First, within-run and between-run precision were determined using three control samples (200, 400, and 900 ng/mL) tested over a two-week period. Within-run coefficients of variation (CV) for the three controls were 1.4, 2.2, and 2.5%, respectively; the between-run CVs were 2.5, 3.1, and 4.0%, respectively. The cross-reactivity with norpropoxyphene, the major metabolite of propoxyphene, was concentration dependent and in the range of 29.3 to 92.6%. Propoxyphene and its metabolite were assayed in biological samples the same day using the Abbott TDx and GC/MS. Tissue preparations were analyzed by TDx without specimen pretreatment other than homogenization and dilution with saline. The TDx results were in accordance with the results obtained by GC/MS.

The determination of propoxyphene, norpropoxyphene, and methadone in postmortem blood and tissues by high-performance liquid chromatography.

This paper describes the quantitative analysis of propoxyphene (PPX), its major metabolite, norpropoxyphene (NPPX), and methadone (METH) in blood and tissue specimens taken at autopsy in cases of suspected drug ingestion. Specimens are extracted into an organic solvent, back-extracted into acid, then reextracted into organic solvent and evaporated to dryness. The reconstituted extracts are subjected to analysis by reversed-phase ion-pair chromatography. The method is linear from 0.1 to 10 mg/L. Recoveries from blood are 86%, 93%, and 91% for PPX, NPPX, and METH respectively. Within-run coefficients of variation are 4.5%, 4.8%, and 2.6% and day-to-day coefficients of variation are 4.7%, 6.8%, and 3.7% for PPX, NPPX, and METH respectively at 1.0 mg/L for each drug.

Evaluation of the online immunoassay for propoxyphene: comparison to EMIT II and GC-MS.

A study was conducted to compare the clinical sensitivity of the OnLine and EMIT II assays for propoxyphene (PPX) use in human urine. A total of 5138 random clinical samples were evaluated by both OnLine and EMIT II. Samples that were positive for each immunoassay were confirmed for PPX and norpropoxyphene (NPPX) by gas chromatography-mass spectrometry (GC-MS). There were 14 samples that were identified positive by both immunoassays and confirmed positive by GC-MS. An additional six samples were positive by OnLine, negative by EMIT II, and confirmed positive by GC-MS. There was one unconfirmed positive sample identified by each immunoassay, and 5116 samples were identified as negative by both immunoassays. The increased sensitivity by OnLine can be attributed to the cross reactivity of the OnLine antibody, which is higher than the cross reactivity of the EMIT II antibody for NPPX (77% versus 7%, respectively). The high concentrations of NPPX, relative to those of PPX, found in all of the clinical samples suggest that laboratories that currently confirm for PPX should confirm for NPPX in order to obtain a better correlation between immunoassay results and GC-MS confirmations.

An apparent fatal valproic acid poisoning.

While extensive data on therapeutic concentrations of valproic acid in plasma exist, data on toxic or fatal levels are very limited and superficial, interpretation of isolated blood levels obtained post-mortem is therefore a difficult task. A case of sudden death is described here. The circumstances are known and strongly indicate a fatal, suicidal poisoning by valproic acid at a dose of 56.4 g. Valproic acid was quantified in tissues by gas chromatography, and its identity confirmed by gas chromatography/mass spectrometry. The tissue concentrations found post-mortem were as follows: blood 720 mg/L; liver 800 mg/kg; stomach contents 1700 mg. Phenobarbitone and propoxyphene were also present at therapeutic concentrations. This data should provide a useful reference for interpretive purposes.

Respirator toxicology.

A case of delayed death resulting from drowning was investigated. A disparity in the propoxyphene concentrations determined in antemortem and postmortem specimens appeared to exist. The disparity is apparently a product of the fact that cardiopulmonary function had been maintained artificially for 57 h, with subsequent tissue autolysis that was demonstrated microscopically. Interpretation of laboratory findings on postmortem specimens must be done with caution when the interval between drug exposure and death has been interrupted by respiratory therapy.

Identification of drugs and their derivatives.

This paper represents a brief review of traditional procedures for the analysis of confiscated drugs and the application of these procedures to the products formed after reductive fragmentation and halogen-acetylated derivatization of the drugs. Confiscated pills, already tentatively identified by some previous procedure, were analyzed by the use of infrared spectroscopy, ultraviolet spectroscopy, and gas chromatography. The same drugs were confirmed by analyzing the alcohols obtained after reductive fragmentation with LiA1H4 as well as the acetylated derivative fractions. Infrared spectra were determined from salt plate films after the evaporation of acidic or basic chloroform extracts of the pill; similarly, ultraviolet spectra were obtained by processing each drug in acidic and basic aqueous solutions. Gas chromatographic analyses involved the determination of characteristic retention times as a function of temperature and the preparation of, and determination of retention times for, derivatives of the drugs. No procedures were specifically included to remove filler material, save its sparing solubility in the extracting solvents; likewise, no special attempts were made to perfect special microtechniques which would allow all analyses to be performed on a single pill. Desired, rather, were the ease, simplicity, and relative speed realized from analysis of approximately five confiscated pills.

Propoxyphene in postmortem toxicology 1976-1978.

A total of 1859 cases provides the basis for this study in which propoxyphene, and often its major metabolite, was demonstrated by toxicological analysis in the blood or tissues of the deceased at 27 medical examiner or coroner's offices across the United States and Canada. The study period includes the last five months of 1975 through December 1978. The cases describe a clearly defined adult population with a marked tendency toward hypochondria, chronic minor illness, and severe psychiatric problems. The high proportion of suicides (44.1% of the total cases and 54.0% of the drug-caused deaths) and multiple-drug toxicities (88.6%) suggests that the involvement of propoxyphene in many of these fatalities may be of less significance than the phenomenon of "polypharmacy" and self-medication without appropriate medical supervision. This evaluation of propoxyphene provides no evidence that propoxyphene is responsible for "street-drug" fatalities. Its appearance in postmortem toxicological examinations has been declining sharply since 1977, but it continues to be dangerous when used excessively, particularly in combination with alcohol and other central nervous system depressant drugs.

Skeletal muscle as an alternative specimen for alcohol and drug analysis.

In a random group of medical examiner cases, muscle tissue, as well as blood and vitreous humor, was analyzed for ethyl alcohol, and the results were compared. When the blood concentration was greater than 0.10 g/dL, the muscle to blood ratio was 1.00 or less (average 0.94), and when the blood concentration was less than 0.10 g/dL, this ratio was greater than 1.00 (average 1.48). The author proposes that this ratio is dependent upon the time course of absorption and distribution, as has been observed for vitreous humor, but with a more rapid equilibration. Muscle tissue was also analyzed in another group of cases found to be positive for one or more drugs in blood. The concentrations of the drugs in muscle varied from none detected to 6.5 times those in blood and seemed to be dependent on the time course between ingestion and death, as well as on the nature of the drug. For most common basic drugs, the ratios were often near unity. Muscle is proposed as a useful alternative specimen to postmortem blood.