Journal of Analytical Toxicology

Moody, David E.; Laycock, John D.; Spanbauer, Alan C.; Crouch, Dennis J.; Foltz, Rodger L.; Josephs, Jonathan L.; Amass, Leslie; Bickel, Warren K.

doi: 10.1093/jat/21.6.406pmid: 9323518

Buprenorphine is used for the management of pain and has been advocated for the treatment of opioid addiction. Therapeutic doses result in low plasma concentrations of buprenorphine. In order to assess the safety and efficacy of buprenorphine, sensitive analytical methods are needed. Until recently, gas chromatography-positive ion chemical ionization mass spectrometry (GC-PCI-MS) offered the most sensitive method to selectively quantitate buprenorphine. We have developed and validated a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method for buprenorphine. The method is described and compared with a GC-PCI-MS method validated in this laboratory. One-milliliter aliquots of plasma are required for the LC-ESI-MS-MS method and 2-mL aliquots for the GC-PCI-MS method. Buprenorphine-d4 is used as internal standard for both methods. Derivatization with pentafluoropropionic acid anhydride is used for the GC-PCI-MS method, in which the derivatized protonated molecular ions after loss of water are monitored at m/z 596 and 600. For LC-ESI-MS-MS, the parent protonated molecule ions are monitored at m/z 468 and 472. A single-step extraction of basic plasma with n-butyl chloride provided recoveries of 70–87%. Although a limit of quantitation (LOQ) of 0.1 ng/mL could be established for LC-ESI-MS-MS, we could only achieve an LOQ of 0.5 ng/mL with the GC-PCI-MS assay. The GC-PCI-MS method has a linear range of 0.5 to 40 ng/mL (mean r2 = 0.998, n = 7). For quality control samples at 1.0, 2.5, and 12.5 ng/mL, the intra- and interassay coefficients of variation (CV) did not exceed 14%, and percent of targets were within 16%. The LC-ESI-MS-MS method had a linear range of 0.1 to 10 ng/mL (mean r2 = 0.999, n = 7).

Crifasi, Joseph A.; Le, Nha X.; Long, Christopher

doi: 10.1093/jat/21.6.415pmid: 9323519

A sensitive method for the quantitation of fluoxetine and norfluoxetine in biological samples was developed. Blood, urine, and tissue samples were alkalinized and extracted with N-butyl chloride. The extracts were derivatized with pentafluoropropionic anhydride before gas chromatography—mass spectrometry (GC-MS). Selected ions were monitored at m/z 117 and 294 for fluoxetine; m/z 117, 176, and 280 for norfluoxetine; and m/z 122 and 299 for the internal standard fluoxetine-d5. The within-run and between-run precision as well as recovery were determined for both analytes. The empirical limit of detection was determined to be 12.5 µg/L for both fluoxetine and norfluoxetine, whereas the empirical limit of quantitation was 25 µg/L for both drugs. Calibration curves were linear in the range of 50–1000 µg/L for both analytes. Some drugs that were known or suspected of interfering with high-performance liquid chromatography and GC methods for fluoxetine and norfluoxetine were tested for interference. This is the only reported method that combines the use of a deuterated internal standard, selected ion monitoring by GC-MS, and derivatization for the identification and quantitation of fluoxetine and norfluoxetine.

Wilkins, Diana G.; Valdez, Angelique S.; Krueger, Gerald G.; Rollins, Douglas E.

doi: 10.1093/jat/21.6.420pmid: 9323520

A sensitive and specific method was developed for the quantitative analysis of I-α-acetylmethadol (LAAM), I-α-acetyl-N-normethadol (norLAAM), and I-α-acetyl-N,N-dinormethadol (dinorLAAM) in hair. In the development of this method, it was determined that sample pretreatment methods performed by the laboratory greatly affect the measured concentrations of drug and metabolite in hair. Deuterated internal standards were added to 20-mg hair samples and the samples digested overnight in a buffered solution of Protease Type VIII enzyme. Digests were extracted by modification of a liquid-liquid extraction procedure developed previously in our laboratory for the analysis of plasma and tissues. Derivatized extracts were analyzed on a Finnigan MAT® 4500 mass spectrometer in positive ion chemical ionization mode using methane and ammonia reagent gases, helium carrier gas, and a DB-5MS (30 m, 0.25-µm film thickness) capillary column. The assay was linear to 50 ng/mg hair (r = 0.99) for all three compounds with a limit of quantitation experimentally determined to be 0.5 ng/mg for LAAM and 0.3 ng/mg for norLAAM and dinor/AAM. Intra-assay precision ranged from 1.0 to 10.5% for the three analytes at concentrations of 0.5, 5.0, and 25.0 ng/mg of hair. Interassay precision ranged from 4.7 to 12.9%. The performance of the method was also evaluated for its utility in detecting and quantitating LAAM, norLAAM, and dinorLAAM in hair from rats (n = 6) that had been administered 3 mg/kg LAAM intraperitoneally once daily for five days. LAAM, norLAAM and dinorLAAM were detectable in pigmented hair at concentrations of 1.27 ng/mg (± 0.04), 1.28 ng/mg (± 0.014), and 2.89 ng/mg (± 0.014), respectively. Five laboratory wash solvents were then evaluated for their effect on the measured concentration of LAAM and metabolites in the rat hair. Phosphate buffer and 1% SDS washes substantially reduced the measured LAAM, norLAAM, and dinorLAAM concentrations by at least 30%, which suggests that drug incorporated into hair is removed (extracted) during the laboratory wash procedures. Wash procedures using methanol, methylene chloride, or water reduced the measuredconcentrations by no more than 20%. Because measured concentrations of LAAM, norLAAM, and dinorLAAM in hair appear to depend on the specific wash procedures used by a laboratory, quantitative data must be interpreted cautiously based on the sample pretreatment conditions.

O'Neal, Carol L.; Poklis, Alphonse

doi: 10.1093/jat/21.6.427pmid: 9323521

In addition to 6-monoacetylmorphine (6-MAM), acetylcodeine (AC) has been suggested as a marker for the use of illicit heroin. We report a sensitive opiate gas chromatographic-mass spectrometric assay that detects AC, diacetylmorphine, and the propionylated derivatives of codeine, morphine, 6-MAM, and norcodeine. The analytes were extracted by solid phase with recoveries from 62 to 98%. The limits of detection (LOD) and quantitation (LOQ) for AC was 0.5 and 1.0 µg/L. The LOD of the other analytes was 2.0 µg/L and the LOQs ranged from 2 to 10 µg/L. The assay was linear for each analyte from the LOQ to 200 µg/L or 400 µg/L (morphine and codeine) with r ≥ 0.996, except for diacetylmorphine which was linear to 100 µg/L with r = 0.994. The within-run and between-run precision were below 10% CV for all analytes. There was no significant hydrolysis of AC to codeine in urine (pH 4.7 and 8.0) after 23 weeks of refrigeration or freezing. After storage at room temperature in urine of pH 8.0, AC was completely hydrolyzed after 5 weeks, but at pH 4.7, 58% of the AC remained after 15 weeks of storage at room temperature. The sensitivity of this assay was adequate to detect AC in the urine of heroin abusers. In preliminary studies, AC was detected in 6 of 69 opiate positive urines. Concentrations ranging from 1 to 48 µg/L were observed. These concentrations were found to be low when compared with the concentrations of 6-MAM, codeine, and morphine also detected in the urines.

O'Dell, Lisa; Rymut, Kathy; Chaney, Glynn; Darpino, Tony; Telepchak, Michael

doi: 10.1093/jat/21.6.433pmid: 9323522

A sensitive and reliable method was developed for the identification and quantitation of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) in urine using a microbed solid-phase extraction (SPE) column. Extensive method validation is presented that evaluates the recovery, linearity, precision, limit of quantitation, limit of detection, and capacity of THCCOOH in urine extracts using CLEAN SCREEN®,1 reduced solvent volume (RSV) SPE columns followed by gas chromatographic-mass spectrometric analysis. The mean recovery of THCCOOH at concentrations of 2, 3, 5, 15, 50, and 150 ng/mL was found to be 91% with coefficients of variation of 7.3% or less. Linearity of the method ranged from 1.95 to 1000 ng/mL with sensitivity at 1 ng/mL for THCCOOH. The sorbent was found to retain at least 1000 ng/mL of THCCOOH with no analyte breakthrough using the described method. Reduction in both processing times and total solvent volume is shown. The RSV SPE columns showed excellent efficiency and performance while demonstrating recoveries, cleanliness, and dynamic ranges comparable with standard SPE products.

McCambly, Kristina; Kelly, Raymond C.; Johnson, Ted; Johnson, James E.; Brown, W. Craig

doi: 10.1093/jat/21.6.438pmid: 9323523

We have evaluated the use of the Hamilton Microlab 2200 robotic pipetting system modified to conduct solid-phase extractions of amphetamines from urine. The Hamilton system is a programmable XYZ robotic sample handling instrument compatible with commercial solid-phase extraction (SPE) columns in the most commonly available sizes. During the extraction and elution steps, the system delivers programmable positive pressure with pressure controlled feedback so as to ensure consistent recovery. The system increases sample throughput while reducing technician hands-on time and improving sample-to-sample and batch-to-batch consistency. In comparison with the manual SPE method, the automated scheme provides similar analyte recovery, accuracy, and precision and a reduced potential for laboratory errors. The method's upper limits of linearity, detection, and Iquantitation were, respectively, 10,000, 100, and 100 ng/mL for amphetamine and 25,000, 50, and 50 ng/mL for methamphetamine. Extraction recoveries for the compounds ranged from 88 to 101%. Carryover amounted to less than 0.02% even at 50,000 ng/mL concentrations of analyte. A typical automated run required 20 min of technician time versus 90 min for a corresponding manual SPE procedure. The automated procedure proved to be a reliable and labor-efficient addition to the laboratory.

Fitzgerald, Robert L.; O'Neal, Carol L.; Hart, Bradley J.; Poklis, Alphonse; Herold, David A.

doi: 10.1093/jat/21.6.445pmid: 9323524

Recent innovations in mass spectrometry (MS) have led to the development of instruments with increased capabilities, smaller footprints, and relatively low cost. The traditional MS in most toxicology laboratories is a quadrupole system equipped with electron impact ionization. Recently, an ion trap with electron impact, positive chemical ionization, negative chemical ionization, and tandem MS capabilities was introduced by Finnigan MAT. This paper compares the sensitivity and precision of ion-ratio measurements between a Finnigan GCQ ion-trap mass spectrometer (ITMS) and a Hewlett Packard quadrupole mass spectrometer (QMS) using electron impact ionization with diazepam as the model compound. Additionally, the sensitivity and precision of ion ratio measurements are evaluated for the ITMS using positive chemical ionization, negative chemical ionization and tandem MS modes of analysis. In the full scan mode (m/z 50–650, 1 Hz), the ITMS had an average signal-to-noise ratio (S/N) of 1400 for a 2-ng injection of diazepam (10 injections per day for 5 days), within-run ion ratio precision had coefficients of variation from 5 to 11%. Using similar full scan conditions, a 10-ng injection of diazepam on the QMS had an average S/N ratio of 160, and precision of ion ratio measurements varied from 5 to 13%. In the selected ion mode (SIM) of analysis (three ions, 2 Hz), the ITMS had an average S/N of 14,000 for a 2-ng injection and ion-ratio precision ranging from 6 to 15%. Using similar SIM conditions, a 2-ng injection in the QMS had an average S/N of 3000 with ion ratio standard deviations of 0.67 to 2.9%. Overall, the ITMS provided at greater S/N, equivalent precision in full scan, but was 5- to 10-fold less precise in measuring ion ratios in the SIM mode as compared with the QMS.

Fogerson, Robert; Schoendorfer, Don; Fay, John; Spiehler, Vina

doi: 10.1093/jat/21.6.451pmid: 9323525

Sweat was collected with the PharmChek™ sweat patch, and drugs were eluted from the collection pad of the patch. A solid-phase enzyme immunoassay (EIA) using microtiter plates was modified for the analysis of opiates in sweat. After opiate administration, sweat contains primarily parent opiate (heroin, codeine) and lipophilic metabolites (6-monoacetylmorphine [6-MAM]). The immunoassay was determined to have a cross-reactivity with codeine of 588%, with hydrocodone of 143%, with diacetylmorphine of 28%, and with 6-MAM of 30% relative to 100% for the morphine calibrators. The optimum cutoff concentration for this modified assay was determined by receiver operator characteristic analysis using 215 patches from 95 subjects to be 10 ng/mL morphine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 86.9% and a diagnostic specificity of 92.8% versus gas chromatography-mass spectrometry (GC-MS), which was the reference method. The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 7.8%, and the interassay coefficient of variation (CV) was 39%. Analysis of spiked patches around the cutoff gave a percent positive threshold of approximately 50% between 10 and 15 ng/mL and a 95% confidence level for a positive result by the EIA between 20 and 25 ng/mL. Eighteen possible adulterants that could be injected into or under the patch were studied. Two (tile cleaner and detergent) can cause false-positive responses in the immunoassay. Two adulterants reduced response to spiked drug (Visine eye drops and Ben Gay ointment), which could cause a false-negative response. All results were 100 confirmed by GC-MS. The clinical sensitivity and specificity for detecting drug use by analyzing sweat collected from human subjects following known doses of codeine (0, 30, and 60 mg orally) or heroin (20 mg intravenously) were 76 and 100%, repectively.

Cody, John T.; Valtier, Sandra

doi: 10.1093/jat/21.6.459pmid: 9323526

Screening large numbers of urine samples for drugs of abuse is typically accomplished using immunoassays that allow for processing large numbers of samples without the requirement of sample preparation before analysis. Until fairly recently, screening of lysergic acid diethylamide (LSD) in urine samples could only be accomplished by the use of radioimmunoassays (RIA). Recently, new nonisotopic immunoassays have been developed for the screening of samples for LSD. These assays lend themselves to rapid, high-volume, automated analysis compared with RIA procedures. In order to evaluate the current commercially available assays, samples prepared at known concentrations were tested by each of the assays. In addition, samples from known use of LSD were tested and the performance of each of the assays compared. The assays examined in this study included RIA assays from Roche Diagnostics (Abuscreen) and Diagnostic Products (coat-a-count) and nonisotopic assays from Roche (OnLine), Behring (EMIT), Boehringer Mannheim (CEDIA), and STC (Microplate EIA). Assays that could readily be carried out in a semiquantitative mode (determining concentration based on a calibration curve) were evaluated as to their relative response to the samples tested. All of the assays evaluated identified all of the samples which confirmed positive by gas chromatography—mass spectrometry (GC-MS). Likewise, each of the assays identified some samples which did not confirm as positive by GC-MS.

Cone, Edward J.; Oyler, Jonathan; Darwin, William D.

doi: 10.1093/jat/21.6.465pmid: 9323527

Saliva concentrations of cocaine, benzoylecgonine, ecgonine methyl ester, and anhydroecgonine methyl ester were measured by gas chromatography—mass spectrometry in six healthy male subjects following cocaine administration by the intravenous, intranasal, and smoked routes of administration. Cocaine appeared in saliva rapidly following all routes of administration. Saliva/plasma (S/P) ratios were generally greater than 1, and there was evidence of moderate to extreme contamination of saliva by cocaine immediately following intranasal and smoked routes of administration. Contamination of the oral cavity and saliva cleared rapidly. Saliva obtained 2 h after dosing appeared to be free of contamination and demonstrated S/P ratios comparable with intravenous administration. Benzoylecgonine and ecgonine methyl ester concentrations were consistently low and were only comparable with cocaine concentrations at times when cocaine concentrations had declined to below 100 ng/mL. Anhydroecgonine methyl ester was detectable in saliva following smoked drug administration, but it was quickly cleared. Terminal half-life estimates for cocaine administered by the intranasal and smoked routes were significantly shorter in saliva compared with those measured in plasma. Half-life estimates following intravenous administration tended to be lower for saliva than plasma, but the differences were not significant. The duration of pharmacologic effects was generally the same as or shorter than detection times of cocaine in plasma and saliva. Overall, the study demonstrated the usefulness of saliva as a test matrix for the detection and measurement of cocaine following administration by different routes of administration.