Journal of Analytical Toxicology

Fritch, Dean; Blum, Kristen; Nonnemacher, Sheena; Haggerty, Brenda J.; Sullivan, Matthew P.; Cone, Edward J.

doi: 10.1093/jat/33.9.569pmid: 20040131

Analytical methods for measuring multiple licit and illicit drugs and metabolites in oral fluid require high sensitivity, specificity, and accuracy. With the limited volume available for testing, comprehensive methodology is needed for simultaneous measurement of multiple analytes in a single aliquot. This report describes the validation of a semi-automated method for the simultaneous extraction, identification, and quantitation of 21 analytes in a single oral fluid aliquot. The target compounds included are amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, pseudoephedrine, cocaine, benzoylecgonine, codeine, norcodeine, 6-acetylcodeine, morphine, 6-acetylmorphine, hydrocodone, norhydrocodone, dihydrocodeine, hydromorphone, oxycodone, noroxycodone, oxymorphone, and phencyclidine. Oral fluid specimens were collected with the Intercept® device and extracted by solid-phase extraction (SPE). Drug recovery from the Intercept device averaged 84.3%, and SPE extraction efficiency averaged 91.2% for the 21 analytes. Drug analysis was performed by liquid chromatography-tandem mass spectrometry in the positive electrospray mode using ratios of qualifying product ions within ±25% of calibration standards. Matrix ion suppression ranged from −57 to 8%. The limit of quantitation ranged from 0.4 to 5 ng/mL using 0.2 mL of diluted oral fluid sample. Application of the method was demonstrated by testing oral fluid specimens from drug abuse treatment patients. Thirty-nine patients tested positive for various combinations of licit and illicit drugs and metabolites. In conclusion, this validated method is suitable for simultaneous measurement of 21 licit and illicit drugs and metabolites in oral fluid.

Fernández, María del Mar Ramírez; Wille, Sarah M.R.; Samyn, Nele; Wood, Michelle; López-Rivadulla, Manuel; De Boeck, Gert

doi: 10.1093/jat/33.9.578pmid: 20040132

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur® Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 µL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two ‘in house’ QCs for blood and urine, respectively. Responses were linear over the investigated range (r2 > 0.99, 2.5–400 µg/L for blood and 25–1000 µg/L for urine). Limits of quantification were determined to be 2.5 and 25 µg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 µg/L for blood and 0.25 to 2.5 µg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 µg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

He, Lei-ping; Di, Bin; Du, Ying-xiang; Yan, Fang; Su, Meng-xiang; Liu, Hua-qing; You, Lin-jun

doi: 10.1093/jat/33.9.588pmid: 20040133

A rapid, sensitive, and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method was developed and validated for simultaneous determination of aconitine (AC), mesaconitine (MA), and hypaconitine (HA), the three toxic constituents from Sini decoction (SND) in rat plasma. After the addition of citalopram as the internal standard (IS), plasma samples were basified with 100 µL 10% ammonium hydroxide, and then extracted with 1 mL ethyl acetate. Chromatographic separation was performed on a CN column (250 mm × 4.6 mm, 5 µm) with a mobile phase of methanol/40 mM ammonium acetate/formic acid (950:45:5, v/v/v) at the flow rate of 1.0 mL/min. Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with positive mode. The method was validated over the concentration ranges of 0.01–10 ng/mL for AC, MA, and HA. The variation coefficients were always < 15% for both intraday and interday precision for each analyte. Mean accuracies were also within ±15%. The method was proved to be sensitive, rapid, specific, accurate, and reproducible. It has been successfully applied to the pharmacokinetics study on rats after oral administration of SND.

Dirikolu, L.; Chakkath, T.; Fan, T.; Mente, Nolan R.

doi: 10.1093/jat/33.9.595pmid: 20040134

In veterinary medicine, lomustine has been successfully used primarily for the treatment of resistant lymphoma and also for the treatment of mast cell tumors, intracranial meningioma, epitheliotropic lymphoma, and histiocytic sarcoma in dogs either alone or in combination with other chemotherapeutic agents. Even though lomustine is commonly used in dogs primarily for the treatment of resistant lymphoma, there is no pharmacokinetics information available regarding this compound in dogs. In the present study, we developed and validated a simple high-performance liquid chromatography (HPLC) method with a one-step liquid-liquid extraction procedure to detect and quantify lomustine and its two monohydroxylated metabolites (trans- and cis-4′-hydroxylomustine) in canine plasma for future pharmacokinetic studies. The HPLC-diode-array detection method reported here readily detects lomustine, cis-4′-hydroxylomustine, and trans-4′-hydroxylomustine in canine plasma with a limit of detection of lomustine, cis-4′-hydroxylomustine, and trans-4′-hydroxylomustine in plasma of about 10 ng/120 µL, 5 ng/120 µL, and 5 ng/120 µL, respectively. The mean extraction efficiency values for lomustine, cis-4′-hydroxylomustine, and trans-4′-hydroxylomustine were 73%, 90%, and 89%, respectively, from canine plasma samples on HPLC. The present study also provides stability information about lomustine and its two monohydroxylated metabolites in canine plasma and methanol solution stored at various conditions.

Björnstad, Kristian; Helander, Anders; Hultén, Peter; Beck, Olof

doi: 10.1093/jat/33.9.604pmid: 20040135

Preparations of the plant Acorus calamus (calamus or sweet flag) (A. calamus) are available via internet trade and marketed as being hallucinogenic. In 2003–2006, the Swedish Poisons Information Centre received inquiries about 30 clinical cases of intentional intoxication with A. calamus products. The present investigation aimed to identify α- and β-asarone, considered active components of A. calamus, and metabolites thereof in urine samples collected in seven of these cases. To further aid the identification of asarone biotransformation products, a calamus oil preparation was incubated with the fungus Cunninghamella elegans, which is used as a microbial model of mammalian drug metabolism. Using gas chromatography-mass spectrometry (GC-MS) analysis in selected ion monitoring mode, α-asarone was detected in five urine samples at concentrations ranging between ∼ 11 and 1150 µg/L and β-asarone in four of those at ∼ 22–220 µg/L. A previously identified asarone metabolite, trans-2,4,5-trimethoxycinnamic acid (trans-TMC), was detected in the fungus broth by liquid chromatography-tandem mass spectrometry whereas cis-TMC was tentatively identified in the human urine samples. Using GC-MS, a hydroxylated asarone metabolite was identified both in fungus broth and urine samples. However, this study demonstrated no evidence for the presence of 2,4,5-trimethoxyamphetamine, claimed as a hallucinogenic component of A. calamus. The main clinical symptom reported by the patients was prolonged vomiting that sometimes lasted more than 15 h.

Westin, Andreas A.; Huestis, Marilyn A.; Aarstad, Kjell; Spigset, Olav

doi: 10.1093/jat/33.9.610pmid: 20040136

Differentiating new intake of drugs-of-abuse from residual drug excretion may be difficult, especially following chronic drug usage and for drugs with long elimination half-lives such as cannabis. In the present case, cannabis was found in the urine of a young pregnant woman following heavy and chronic cannabis use. She was warned that if she continued using cannabis while pregnant she would be forced to be hospitalized. She was subjected to serial urine testing with 2–7-day intervals. Urinary 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) concentrations, measured by liquid chromatography-mass spectrometry, declined from 348 to 3.9 ng/mL over a surprisingly long period of 12 weeks (84 days). Several algorithms for detecting new drug intake were applied during this time course; most indicated that the woman continued to smoke cannabis following the first urine test. The woman denied any use after the first specimen collection. In retrospect, her THCCOOH excretion profile supports her story. Algorithms for detecting new drug intake have been validated for occasional cannabis users only. We advise caution when interpreting urine test results from heavy, chronic cannabis users, especially when serious consequences are involved.

McIntyre, Iain M.; Sherrard, James L.; Nelson, Craig L.

doi: 10.1093/jat/33.9.615pmid: 20040137

There has been an increased awareness of illicit opiate abusers using the narcotic oxymorphone (Opana®) by inhalation. Many laboratory screening techniques currently in use cannot detect oxymorphone in blood or urine. Consequently, biological specimens containing low to moderate concentrations of oxymorphone will likely go undetected. The circumstances, pathology findings, and toxicology results of two fatalities involving oxymorphone are presented. An opiate confirmation gas chromatography-mass spectrometry (GC-MS) procedure, described in detail was able to detected, confirm, and quantify oxymorphone in both subjects. The blood concentrations were 0.05 mg/L (50 µg/L) and 0.12 mg/L (120 µg/L).

Raymon, Lionel P.; Isenschmid, Daniel S.

doi: 10.1093/jat/33.9.620pmid: 20040138

Jones, A.W.

doi: 10.1093/jat/33.9.623pmid: 20040139