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Stability is an important consideration in the use of specimens for accurate determination of analyte concentrations. To determine the long-term stability for analytes routinely analyzed by mass spectrometry in this laboratory, quality-control (QC) results were plotted versus time. The time required for the initial concentration to reach a specified level of deviation (i.e., 15%) was then determined from the slopes. QCs were prepared at 1-3 concentrations in drug-free matrix and stored at approximately -20 degrees C; urines were fortified with 1% sodium fluoride; plasmas (except for cocaines) were prepared with heparin. For cocaine and metabolites, the plasma was either fortified with 2% sodium fluoride or with 2% sodium fluoride and 1 mg% physostigmine after adjustment of the plasma pH to 6.0. In urine, amphetamine, methamphetamine, codeine, morphine, benzoylecgonine (BZE), and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) slopes did not exceed a 15% deviation before 852 days. Cocaine, however, reached a 15% reduction at 165 days. When cocaine and BZE were prepared in plasma with just 2% sodium fluoride, negative slopes reached 15% deviation within 154 and 111 days, respectively. Further fortification with physostigmide and adjustment of the pH extended this time frame significantly. Delta9-Tetrahydrocannabinol (THC) and THCA in plasma had negative slopes that deviated by 15% just prior to one year. l-Alpha-acetylmethadol (LAAM), methadone, and their N-demethylated metabolites in urine did not have any negative slopes exceeding 15% before 686 days. Several of the compounds had positive slopes. Those for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine reached the 15% mark within 98 days. Those for LAAM, norLAAM, and dinorLAAM were concentration dependent. The 25-ng/mL controls reached 15% at 158-216 days. The 700-ng/mL controls reached 15% at 784-1340 days. In plasma, only naltrexone and buprenorphine displayed negative slopes at all three concentrations, reaching the 15% mark as early as 576 and 272 days, respectively. LAAM, norLAAM, dinorLAAM, ibogaine, 6-beta-naltrexone, risperidone, and 9-OH-risperidone did not exceed a 15% deviation before 416 days. To attempt to validate this method, two sets of clinical plasma samples that had been analyzed for buprenorphine were reanalyzed 644 and 869 days after the initial analyses. Those reanalyzed after 644 days were not statistically different from initial analyses, whereas those stored for 869 days were statistically different (p < 0.05). As the average time to reach 15% deviation for the three concentrations of buprenorphine QCs was 782 days, this suggests that extrapolation of QC results gathered over time may provide a reliable method to estimate long-term stability limits for drugs stored under the same conditions as the QC samples.

Immunoassays are commonly used to screen samples prior to confirmation by gas chromatography-mass spectrometry (GC-MS). This serves two purposes: it provides a second method for positive samples, and it allows exclusion of negative samples from further confirmatory testing. In addition, immunoassay results can be used in some cases to determine if dilution of the sample will be required during the confirmatory assay. We used 878 sweat patches worn by 38 subjects receiving treatment for cocaine dependence to compare analysis of the extracts of the patches for cocaine immuno-equivalents by radioimmunoassay (RIA) with determination of cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) by GC-MS. Preliminary validation experiments demonstrate that the GC-MS method using positive ion chemical ionization had sufficient specificity and recovery to support a lower limit of quantitation (LLOQ) of 4 ng/patch and was precise and accurate across a linear range up to 500 ng/patch. Cocaine ranging from the LLOQ to 31,900 ng/patch was found in 660 of the samples; BE ranging from the LLOQ to 3470 ng/patch was found in 530 of the samples; and EME ranging from the LLOQ to 2280 ng/patch was found in 476 of the samples. In a subset of 238 samples semiquantitative use of the RIA gave results that agreed with GC-MS with a correlation coefficient of 0.986, but averaged approximately 23% lower. Although this accuracy of the RIA supported its use as a sole quantitative assay, the limited linear range of the RIA (4-200 ng/patch) proved impractical for this purpose. Receiver operator characteristic analysis of the cutoffs of the RIA and GC-MS suggested optimal cutoffs of 5 and 4 ng/patch, respectively. At these cutoffs, the RIA had sensitivity of 90.0% and specificity of 92.2%. For samples that had RIA results greater than the high calibrator (N = 228), various dilution schemes were assessed for their ability to predict retention of either cocaine alone or cocaine and both metabolites within the linear range of the GC-MS. When cocaine was the only analyte of interest, a single 20-fold dilution retained 200 (87.7%) of the samples. This compared to an optimal scheme where a different dilution was selected for each one-tenth ratio (< 0.1, 0.1-0.2, etc.) where 211 (92.5%) of the samples were retained. When trying to retain cocaine and both metabolites, dilution schemes were less successful as BE and EME would often fall below the LLOQ of the GC-MS. A single fivefold dilution of all samples retained 115 (50.4%) compared to an optimum of 143 (62.7%). The optimum could be approached with four dilution sets retaining 142 of the samples. Time expended on performing RIA analysis of all the samples was cost-effective when the results were used to exclude negatives from and predict dilutions required for GC-MS analysis. RIA offers a cost-effective, sensitive, and specific alternative initial test for cocaine determination in extracts of sweat patches.

An accurate and reliable gas chromatographic-mass spectrometric method was developed to analyze tissue, whole blood, plasma, and urine samples for cocaine (COC) and its major metabolites. COC, benzoylecgonine (BZE), and ecgonine methyl ester (EME) were isolated from the biological matrix using solid-phase extraction, and the tert-butyldimethylsilyl derivatives of BZE, EME, and their deuterium-labeled internal standards were formed. Separation of the compounds was performed by capillary chromatography, and analysis was performed by positive ion chemical ionization mass spectrometry using methane and ammonia as the reagent gases. The tert-butyldimethylsilyl derivatives of BZE and EME were stable and produced mass spectral ions with higher mass-to-charge ratios than trimethylsilyl derivatives. Recovery of COC and its metabolites exceeded 80% at all three concentrations tested. Linearity of the method was established from 2.5 to 2000 microg/L. Intra-assay precision had a coefficient of variation (CV) of less than 9% for all analytes when tested at 10, 25, 100, and 200 microg/L. Interassay precision also had a CV of less than 9% for COC, BZE, and EME at 25 and 100 microg/L. At 200 microg/L, %CVs for COC, BZE, and EME were 11.5, 12.0, and 12.7, respectively. In addition to the analysis of COC, BZE, and EME, the method was used to quantitate cocaethylene and to identify norcocaine.