A rapid gas chromatographic method quantitating clozapine in human plasma or serum for the purpose of therapeutic monitoring.

A gas chromatographic method using nitrogen-phosphorus detection was developed to quantitate clozapine in plasma or serum. Methyl clonazepam was used as an internal standard. Sample preparation included a single-step extraction with ethyl acetate, which was injected directly onto a wide-bore capillary column. Within-run and total precision, measured as percent coefficient of variation, were determined at low, therapeutic, and high clozapine plasma concentrations. The within-run precision for the low, therapeutic, and high clozapine plasma samples was 5.2, 2.7, and 2.4%, respectively. The total precision for the low, therapeutic, and high clozapine plasma samples was 10.0, 2.6, and 2.0%, respectively. Analytical accuracy was evaluated by comparing quantitative results with those obtained from a reference laboratory. Those samples containing therapeutic or high concentrations agreed within 3%; the sample containing a subtherapeutic concentration differed by 11.9%. The limit of quantitation was determined to be 35 ng/mL, and the upper limit of linearity was 3000 ng/mL. No significant interferences were detected after testing more than two dozen drugs and metabolites.

A high-performance liquid chromatographic method for quantitating bupropion in human plasma or serum.

We report a high-performance liquid chromatographic (HPLC) procedure for quantitating bupropion in serum or plasma for the purpose of therapeutic monitoring. Bupropion and its internal standard, a fluorinated analogue of bupropion, are extracted into hexane-isoamyl alcohol (96:4) after the addition of 400 microL 0.1N KOH. The organic phase is evaporated, reconstituted with 200 microL acetonitrile, and then analyzed on a silica column using a mobile phase consisting of 95% methanol and 5% NH4H2PO4. The ultraviolet detector is set to monitor 248 nm. Within-run and total precision at a therapeutic concentration of 30 ng/mL are 5.2 and 8.5%, respectively. The lower limit of quantitation is 5 ng/mL, and the upper limit of linearity is 400 ng/mL. More than two dozen drugs and metabolites were tested for interference; fluoxetine was the only analyte demonstrating a retention time that would interfere with bupropion quantitation. Chromatographic analysis time per injection is less than 7 min. This procedure combines a single-step extraction with HPLC analysis to provide rapid and reliable analysis of bupropion.

The reliability of a solid-phase extraction system for the analysis of benzoylecgonine in urine.

Gas chromatographic-mass spectrometric (GC-MS) analysis for benzoylecgonine (BE), a metabolite of cocaine, requires an initial extraction from urine. Although liquid-liquid extraction methods are frequently used, solid-phase extraction (SPE) may be preferable for obtaining reliable results and clean chromatograms. We describe a 12-month study that evaluates the accuracy, precision, variability between analysts, variability between column lots, and cleanliness of BE extracts using SPE columns followed by GC-MS analysis. The overall mean for a control urine sample prepared at 150 ng/mL is 151 ng/mL (N = 293) with a standard deviation of 8.59 and a coefficient of variation (CV) of 5.7%. Within-run precision (measured as CV) at 75, 150, and 2000 ng/mL is 4.0, 1.8, and 0.8%, respectively. Mean results from 10 different analysts vary a maximum of 4.6% from the overall mean of 151 ng/mL, and the CV for 9 out of 10 analysts is 7.0% or less. The CV for the remaining analyst is 10.4%. Quantitative results from nine different lots of SPE columns fluctuate 3.3% from the overall mean of 151 ng/mL, and the CV varies from 3.5 to 6.2%. GC-MS chromatograms following SPE are significantly cleaner (i.e., reduced baseline signal and no interfering peaks) than those from two types of liquid-liquid extractions.

Application of the Technicon Chem 1+ chemistry analyzer to the Syva Emit ethyl alcohol assay in plasma and urine.

The performance of the Technicon Chem 1+ chemistry analyzer with the Syva Emit ethyl alcohol assay in plasma and urine was evaluated. Spiked specimens from 0 to 600 mg/dL were tested, and expected versus measured concentrations were monitored. Linear regression line equations of y = 0.9314x + 5.4 and y = 0.9005x + 4.6, and correlation coefficients (r) of 0.9997 and 0.9995, were obtained for plasma and urine, respectively. A limit of detection of 5 mg/dL for plasma and urine, and a limit of quantitation of 20 mg/dL for plasma and 15 mg/dL for urine were obtained. Recovery was within 10% of expected concentration from 20 to 600 mg/dL. Precision was evaluated, giving the following coefficients of variation: within-run precision: plasma, 1.31-2.20; urine, 1.16-1.21; total precision: plasma, 2.72-3.38; urine, 2.98-4.64. No carry-over was detected when alternating 600 mg/dL and negative specimens. No interference from acetone, isopropanol, or methanol was detected. No significant differences in evaporation of alcohol at two concentrations, or from the two matrices were observed. Evaporation from a small cup (200 microL) was more than twice as great as from a large cup (2 mL). The Chem 1+ was compared to a gas chromatographic method. Plasma specimens of 0-352 mg/dL produced a linear regression line of y = 1.0112x + 6.0, r = 0.9859; urine specimens of 0-313 mg/dL produced a line of y = 1.0493x - 0.3, r = 0.9910. The capability to separate positive and negative specimens at 20% around a cutoff concentration of 20 mg/dL was examined. Four hundred specimens were analyzed, with only one specimen incorrectly classified (a false positive). The Chem 1+ chemistry analyzer demonstrated reliable performance of the Emit ethyl alcohol assay of plasma and urine specimens.

The quantitative conversion of morphine 3-beta-D glucuronide to morphine using beta-glucuronidase obtained from Patella vulgata as compared to acid hydrolysis.

Methods to confirm morphine in urine require hydrolysis to liberate morphine from its 3-beta-D glucuronide (M-3G) conjugate. Lengthy enzyme hydrolysis procedures prolong testing turnaround time whereas rapid enzyme methods may produce a low conversion of M-3G to morphine. The purpose of this study was to evaluate the quantitative conversion of M-3G to morphine in human urine using a thermally stable beta-glucuronidase isolated from Patella vulgata; to compare these findings with those obtained from acid hydrolysis; and to compare between-run imprecision for both hydrolysis methods. We found both enzyme and acid hydrolysis techniques to be efficient, giving 90.4% and 92.8% conversion of M-3G to morphine, respectively. Also, both methods were found to be reproducible. Over a 14 week period, 20 opiate confirmation batches were analyzed by each hydrolysis method; the coefficient of variation for morphine liberated from M-3G was 5.5% for enzyme hydrolysis and 2.7% for acid hydrolysis.

Investigation of interference by nonsteroidal anti-inflammatory drugs in urine tests for abused drugs.

Anecdotal and uncontrolled studies have suggested that nonsteroidal anti-inflammatory drugs produce false-positive results in immunoassay urine tests for some drugs of abuse. This study was performed in 60 volunteers who took ibuprofen as either a single 400-mg dose, or 200 mg three times a day, or 400 mg three times a day, and in 42 patients taking ibuprofen, naproxyn, or fenoprofen in therapeutic regimens for more than 30 days. Of the 510 urines collected from 102 individuals during these dosage regimens, two gave false-positive tests for cannabinoid by enzyme-mediated immunoassay (EMIA), one after 1200 mg of ibuprofen in three divided doses for one day and one in a patient taking naproxyn on a chronic basis; none was falsely positive for benzodiazepines. Two urines were false-positive for barbiturates by fluorescence polarization immunoassay (FPIA), one in a patient taking ibuprofen and one in a patient taking naproxyn. These data, collected prospectively, demonstrate the small likelihood of a false-positive immunoassay test result for cannabinoids, benzodiazepines, or barbiturates after the acute or chronic ingestion of ibuprofen, or after the chronic ingestion of naproxyn or fenoprofen.

An evaluation of the TDX fluorescence polarization immunoassays for procainamide and n-acetylprocainamide.

The TDX fluorescence polarization assays (FPIA) for procainamide (PA) and n-acetylprocainamide (NAPA) were evaluated. Coefficients of variation for within- and between-assay precision studies were less than 6%. Both methods correlated well with a referenced HPLC technique; r2 values for PA and NAPA were 0.980 and 0.986, respectively.

Twin transfusion syndrome: successful in utero treatment with digoxin.

A pregnancy complicated by twin transfusion syndrome is presented. When signs of cardiac failure (edema, ascites and hydramnios) persisted in the recipient twin, maternal digoxin therapy was instituted at 27 weeks' gestation. The signs of failure resolved, and the twins were delivered electively by cesarean section at 34 weeks. At birth, the syndrome was confirmed by examination of the infants and placenta. Both infants survived. Digoxin therapy is recommended for fetal heart failure from circulatory overload in twin transfusion.

Analysis of ethchlorvynol (Placidyl): evaluation of a comparison performed in a clinical laboratory.

Common techniques for analyzing ethchlorvynol (Placidyl) by colorimetry and gas liquid chromatography are evaluated. Matrix effects are thoroughly examined to determine their contribution to the validity of results.

Stability of delta 9-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in blood and plasma.

The stabilities of delta 9-tetrahydrocannabinol (THC) and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol (HO-THC) and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (COOH-THC), were determined in blood and plasma stored at -10 degrees C, 4 degrees C, and room temperature. Each of the cannabinoids was added to freshly-drawn blood and plasma to give concentrations of 20 ng/mL. Two-mL aliquots were stored in silanized tubes and the cannabinoid concentrations were monitored by gas chromatography/mass spectrometry over a 6-month period. No significant changes were observed in the concentrations of the cannabinoids for the first month of storage. However, the concentrations of THC and HO-THC in blood stored at room temperature had decreased significantly at 2 months. No statistically significant changes were detected in cannabinoid concentrations in plasma or blood stored at 4 degrees or -10 degrees C for up to 4 months. After 6 months at room temperature, the blood concentrations of THC and HO-THC had decreased by 90 and 44%, respectively, whereas the concentration of COOH-THC was not significantly different from the control. The possibility of loss of cannabinoids from blood due to adsorption onto the grey stoppers used in Venoject tubes was also investigated. Over a 24-hr period, no significant differences were detected in any of the cannabinoid concentrations regardless of sample size (1.3 or 8 mL), differences in temperature (-10 degrees C, 4 degrees C, or room temperature), or extent of contact with the tube's stoppers.

Formate concentrations in a case of methanol ingestion.

An eight-month-old infant ingested at least 3.5 g of methanol when he accidentally received 5 mL of amoxicillin suspended in 70% methanol. The serum methanol concentration 8 h later was 9.7 mmol/L (310 mg/L) and the formate concentration 23 mmol/L (1.0 g/L). At 18 h after the ingestion, total CO2 had decreased to 6.8 mmol/L. Throughout the second day, 21-32 h after the ingestion, the methanol concentration was 2.8-3.4 mmol/L (90-110 mg/L) and that of formate was 31-33 mmol/L (1.4-1.5 g/L). On the third day, 46 h after the ingestion, methanol was not detected and the formate concentration had declined to 16 mmol/L (720 mg/L). The patient was treated with activated charcoal 7 h after the ingestion and with ethanol, administered both orally and intravenously, 21 h after the ingestion. No abnormalities of the infant's eyes were noted upon ophthalmological examination approximately 55 h after the incident.

An evaluation of the EMIT st assay for the detection of tricyclic antidepressant drugs in plasma or serum.

The EMIT st assay for tricyclic antidepressant drugs (TADs) was evaluated for use as a screening technique in the detection of these drugs in serum or plasma. The qualitative assay was found to be rapid, easy to perform, requires no sample or reagent preparations, and reliably detected the TADs in patient samples at concentrations greater than or equal to 200 ng/mL. The technique also detected seventeen of nineteen patient samples with TAD concentrations ranging from 150-199 ng/mL and ten of forty-three samples with concentrations less than 150 ng/mL. The EMIT st assay was found to be a reliable technique for detecting high concentrations of TADs and is well-suited for use in emergency drug screening situations.

Comparison of quinidine by fluorescence polarization immunoassay and high pressure liquid chromatography.

Analysis of quinidine was performed using fluorescence polarization immunoassay (Abbott TDXTM) with an ion-pairing adsorption high pressure liquid chromatography (HPLC) technique as the comparative method. Correlation of 110 clinical samples was excellent (r2 = 0.983). TDX calibration appeared stable for 14 days. There is minimal contribution to the concentration of quinidine due to the presence of metabolite. The TDX method is rapid, precise, and accurate.

Spurious phenobarbital levels by fluorescence polarization immunoassay using TDx analyzer in patients with renal disease.

Results of phenobarbital analyses are presented for 14 patients with chronic renal failure, and compared using gas-liquid chromatography (GLC), enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA). Correlation of FPIA with GLC and EIA was poor.

An HPLC method for the simultaneous quantitation of quinidine, procainamide, N-acetylprocainamide, and disopyramide.

A high performance liquid chromatographic method is reported, which incorporates three internal standards (I-cinchonidine, N-propylprocainamide, and para-chlorodisopyramide) for the simultaneous quantitation of four commonly prescribed antiarrhythmic drugs: quinidine, procainamide, N-acetylprocainamide, and disopyramide. Compounds were separated using combined ion-pairing and adsorption chromatography on a silica column. Inter-run variation was 5.9 CV% for all drugs.

An evaluation of common methods for acetaminophen quantitation for small hospitals.

Rapid, reliable serum acetaminophen quantitations assist in the diagnosis of the toxic patient and direct the clinician in the course of aggressive treatment. Reliable performance must be tempered with economic consideration, especially for small laboratories with infrequent testing. Two commonly available colorimetric techniques for acetaminophen (nitration and ferric reduction) are evaluated using both commercial and laboratory reagents. Enzyme immunoassay (EMIT) is also performed. Results are compared to an HPLC reference method.

Plasma and urine disposition of 1-alpha-acetylmethadol and its principal metabolites in man.

The disposition of 1-alpha-acetylmethadol (LAAM) in plasma and urine was monitored by GC/CIMS following oral administration of 10 doses (0.73-1.5 mg/kg) over 42 days, to twelve human subjects. Plasma concentration-time course profiles fitted a two-compartment, first order kinetic model. Mean plasma t1/2 alpha for LAAM was 2.4 hours; t1/2 beta was 37.5 hours for the first dose and 46.8 hours for the last dose. The mean terminal half-life for nor-LAAM was 38.2 hours for first and 64.6 for last dose; for dinor-LAAM t1/2 beta was 168 hours, last dose. Drug accumulation occurred in some subjects, but within the study range, dosage was not related to maximum plasma levels nor to accumulation. In urine, the sum of LAAM, nor-LAAM, and dinor-LAAM represented 25% of the dose, and unconjugated methadol metabolites, 1.6-1.7%.

Quantitative liquid-chromatographic technique for the simultaneous assay of tricyclic antidepressant drugs in plasma or serum.

A quantitative "high-pressure" liquid-chromatographic assay for tricyclic antidepressant drugs in plasma or serum is described in which amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and desmethyldoxepin are separated with a 10-micrometer particle size silica column and a methanol/NH4OH/NH4NO3 solvent system. The drugs and two internal standards are extracted with hexane/isoamyl alcohol, the solvent layer is evaporated at 40 degrees C, and the drugs are detected at 254 nm. Drug concentrations are linear with absorbance from 25 to 1000 micrograms/L; within-assay and between-assay CVs are less than or equal to 10%.