Kamisha L. Johnson-Davis

Bupropion increases striatal vesicular monoamine transport

The vesicular monoamine transporter-2 (VMAT-2) is principally involved in regulating cytoplasmic dopamine (DA) concentrations within terminals by sequestering free DA into synaptic vesicles. This laboratory previously identified a correlation between striatal vesicular DA uptake through VMAT-2 and inhibition of the DA transporter (DAT). For example, administration of methylphenidate (MPD), a DAT inhibitor, increases vesicular DA uptake through VMAT-2 in a purified vesicular preparation; an effect associated with a redistribution of VMAT-2 protein within DA terminals. The purpose of this study was to determine if other DAT inhibitors, including bupropion, similarly affect VMAT-2. Results revealed bupropion rapidly, reversibly, and dose-dependently increased vesicular DA uptake; an effect also associated with VMAT-2 protein redistribution. The bupropion-induced increase in vesicular DA uptake was prevented by pretreatment with eticlopride, a DA D2 receptor antagonist, but not by SCH23390, a DA D1 receptor antagonist. We previously reported that MPD post-treatment prevents persistent DA deficits associated with multiple methamphetamine (METH) administrations. Although bupropion attenuated the METH-induced reduction in VMAT-2 activity acutely, it did not prevent the long-term dopaminergic toxicity or the METH-induced redistribution of VMAT-2 protein. The findings from this study demonstrate similarities and differences in the mechanism by which MPD and bupropion affect striatal dopaminergic nerve terminals.

Long-term post-synaptic consequences of methamphetamine on preprotachykinin mRNA expression.

Exposure to repeated high doses of methamphetamine produces long‐term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by ∼20% in the middle striatum and ∼32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by ∼20% in the middle striatum and ∼14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either timepoint. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.

A rapid HPLC method used to establish pediatric reference intervals for vitamins A and E.

BACKGROUND Serum concentrations of the fat-soluble vitamins A (retinol) and E (tocopherol) are measured to assess deficiency and, in the case of vitamin A, toxicity. We modified our existing HPLC method for analyzing vitamins A and E by using a high throughput analytical column and small diameter tubing to reduce analysis time. The modified HPLC method was used to establish pediatric reference intervals for these vitamins. METHODS Serum or plasma proteins were precipitated with ethanol. A and E vitamins were extracted into hexane, evaporated under nitrogen, dissolved in absolute ethanol, and analyzed by HPLC with ultraviolet detection. RESULTS The modified HPLC method correlated well with the existing method. Data analysis from the reference interval study resulted in age-dependent intervals for retinol and non-age-dependent intervals for retinyl palmitate, alpha-tocopherol, and gamma-tocopherol. Gender-based reference intervals were not necessary. CONCLUSIONS We validated a rapid HPLC method for analyzing vitamins A and E that decreased run-time by 60%, mobile phase consumption by 39%, and sample injection volume by 50%. The modified method was used to establish pediatric reference intervals for vitamins A and E in samples from 1136 healthy children aged 7 to 17 y.

Simultaneous quantification of levetiracetam and gabapentin in plasma by ultra-pressure liquid chromatography coupled with tandem mass spectrometry detection.

Introduction: Gabapentin (Neurontin) and levetiracetam (Keppra) are anticonvulsants with novel structures and suggested therapeutic ranges of 2-10 mg/L and 6-20 mg/L, respectively. Gabapentin is also used extensively to manage neuropathic pain, and for this indication, wherein higher doses are prescribed, plasma concentrations of 15-30 mg/L are typical. Objective: Here, we describe a simple rapid assay to support therapeutic drug monitoring of gabapentin and levetiracetam in plasma by ultra-pressure liquid chromatography couples to tandem mass spectrometry (UPLC-MS/MS) detection. Methods: After the addition of internal standard and protein precipitation of patient plasma with methanol:acetonitrile in a 50:50 ratio, 1 μL of supernatant sample is injected onto an Acquity UPLC HSS T3, 1.8 μm, 2.1 × 50 mm (Waters) column. Elution occurs using a linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into a Waters Acquity UPLC TQD, operating in a positive mode to detect gabapentin at transition 172.18 > 154.11, levetiracetam at 171.11 > 126, and internal standard (3-amino-2-naphthoic acid) at 188.06 > 170. Secondary transitions for each analyte are also monitored for gabapentin at 172.18 > 137.06, levetiracetam at 171.11 > 154, and internal standard at 188.06 > 115. Runtime is 1.5 minutes per injection with baseline resolved chromatographic separation. Results: The analytical measurement ranges were 1-150 mg/L for gabapentin and for levetiracetam. Intra-assay imprecision by the coefficient of variance (CV) was less than 8% and interassay CV was less than 5% for both analytes, at 4 different concentrations. Results obtained from patient samples were compared with results generated by established high-performance liquid chromatography-UV methods with the following regression statistics: y = 1.12x − 0.77, r = 0.996, Sy, x = 0.89, and n = 29 for gabapentin and y = 0.991x + 0.70, r = 0.997, Sy, x = 2.24, and n = 30 for levetiracetam. No analytical interferences were identified. Conclusion: In summary, a simple reliable UPLC-MS/MS method was developed and validated for routine clinical monitoring of gabapentin and levetiracetam.

Evaluation of the Abbott ARCHITECT i2000 sirolimus assay and comparison with the Abbott IMx sirolimus assay and an established liquid chromatography-tandem mass spectrometry method.

Background: Sirolimus is indicated for prophylaxis treatment to prevent solid organ rejection. Due to intrapatient and interpatient variabilities in pharmacokinetics, risk of concentration-related toxicity, risk of noncompliance, and a high likelihood of drug-drug interactions, therapeutic drug monitoring of sirolimus is essential. There are several methodologies used clinically to monitor sirolimus, ranging from immunoassay to tandem mass spectrometry, each with potential strengths and limitations. The purpose of our study was to validate the Abbott ARCHITECT i2000 sirolimus assay. Materials and Methods: The Abbott ARCHITECT i2000 sirolimus assay was evaluated for linearity, limit of detection, limit of quantification, imprecision, common interferences, and accuracy. Accuracy was compared with the Abbott IMx sirolimus assay and with an established liquid chromatography-tandem mass spectrometry method. Stability of sirolimus when specimens were stored frozen (−20°C) or refrigerated (2-8°C) and degree of crossreactivity of the i2000 with everolimus were also evaluated. Results: The ARCHITECT i2000 assay demonstrated good linearity, low imprecision, and was free of common interferences. Results for both immunoassay methods were biased slightly high, compared with those of liquid chromatography-tandem mass spectrometry. Sirolimus was stable when samples were stored either refrigerated or frozen. However, the results generated with samples stored refrigerated had an increase in scatter on the regression plots compared with those generated for samples that were stored frozen, indicating that extraction of the drug may be incomplete, particularly with the Abbott IMx assay. In addition, statistical performance indices suggest that the agreement between the ARCHITECT i2000 and Abbott IMx was better for frozen patient samples. The ARCHITECT i2000 and the Abbott IMx methods both exhibited a >100% crossreactivity with everolimus. Conclusions: The Abbott ARCHITECT i2000 instrument demonstrates reasonable sensitivity, precision, and accuracy for supporting routine therapeutic drug monitoring of sirolimus with either refrigerated or frozen whole blood collected from patients who are not comedicated with everolimus.

A Retrospective Analysis of Urine Drugs of Abuse Immunoassay True Positive Rates at a National Reference Laboratory

Urine drug screens are commonly performed to identify drug use or monitor adherence to drug therapy. The purpose of this retrospective study was to evaluate the true positive and false positive rates of one of our in-house urine drug screen panels. The urine drugs of abuse panel studied consists of screening by immunoassay then positive immunoassay results were confirmed by mass spectrometry. Reagents from Syva and Microgenics were used for the immunoassay screen. The screen was performed on a Beckman AU5810 random access automated clinical analyzer. The percent of true positives for each immunoassay was determined. Agreement with previously validated GC-MS or LC-MS-MS confirmatory methods was also evaluated. There were 8,825 de-identified screening results for each of the drugs in the panel, except for alcohol (N = 2,296). The percent of samples that screened positive were: 10.0% for amphetamine/methamphetamine/3,4-methylenedioxy-methamphetamine (MDMA), 12.8% for benzodiazepines, 43.7% for opiates (including oxycodone) and 20.3% for tetrahydrocannabinol (THC). The false positive rate for amphetamine/methamphetamine was ∼14%, ∼34% for opiates (excluding oxycodone), 25% for propoxyphene and 100% for phencyclidine and MDMA immunoassays. Based on the results from this retrospective study, the true positive rate for THC drug use among adults were similar to the rate of illicit drug use in young adults from the 2013 National Survey; however, our positivity rate for cocaine was higher than the National Survey.

A comparison of two FDA approved lamotrigine immunoassays with ultra-high performance liquid chromatography tandem mass spectrometry.

BACKGROUND Lamotrigine is an anti-epileptic drug used as adjunct therapy for seizures. Lamotrigine is commonly used in pregnant women with epilepsy, a population in which therapeutic drug monitoring (TDM) is useful to optimize dose. Drug-drug interactions that can induce or inhibit metabolism or elimination and impaired hepatic function are also possible indications for lamotrigine TDM. Chromatographic techniques are currently used for performing most TDM of lamotrigine, but this may change, as automated immunoassays were recently introduced. METHODS Immunoassays available through Seradyn and ARK Diagnostics were validated using a Beckman AU400e automated chemistry analyzer. The intra-day precision was accessed with 5 replicates of three quality control materials, and inter-assay precision was estimated by assaying the same material over 4 days. Linearity was evaluated by serially diluting a spiked sample and measuring it in duplicate. The 2 methods were compared with ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using 44 authentic patient specimens. RESULTS The intra-day (n=5) and inter-assay (n=20) coefficients of variation were ≤7.5% for the 3 levels tested. The analytical measurement ranges were confirmed as stated by the manufacturers (0 or 1-40 μg/ml). The percent recovery of the quality control materials and Deming regression for the 44 patient results showed good agreement of both immunoassays when compared to the UPLC-MS/MS assay. CONCLUSION The lamotrigine assays studied here produced a slightly lower result than UPLC-MS/MS but were precise and easy to perform.

Demystifying Analytical Approaches for Urine Drug Testing to Evaluate Medication Adherence in Chronic Pain Management

ABSTRACT This comprehensive review of analytical methods used for urine drug testing for the support of pain management describes the methods, their strengths and limitations, and types of analyses used in clinical laboratories today. Specific applications to analysis of opioid levels are addressed. Qualitative versus quantitative testing, immunoassays, chromatographic methods, and spectrometry are discussed. The importance of proper urine sample collection and processing is addressed. Analytical explanations for unexpected results are described. This article describes the scientific basis for urine drug testing providing information which will allow clinicians to differentiate between valid and questionable claims for urine drug testing to monitor medication adherence among chronic pain patients.

Multigene and Drug Interaction Approach for Tamoxifen Metabolite Patterns Reveals Possible Involvement of CYP2C9, CYP2C19, and ABCB1

Tamoxifen is metabolically activated to 4‐hydroxytamoxifen and endoxifen by cytochrome P450 (CYP). CYP phenotypes have been correlated to tamoxifen outcomes, but few have considered drug interactions or combinations of genes. Fewer still have considered ABCB1, which encodes P‐glycoprotein and transports active tamoxifen metabolites. We compared the concentrations of tamoxifen and metabolites in 116 breast cancer patients with predicted phenotypes for CYP2D6, CYP3A4, CYP3A5, CYP2C9, CYP2C19, and ABCB1 genotypes. A significant correlation between CYP2D6 phenotypes and tamoxifen metabolites was seen, strongest for endoxifen (P < .0001). Statistical fit of the data improved when using gene activity scores adjusted for known drug interactions. Concentration of tamoxifen was significantly higher (P = .02) for patients taking a CYP2C19 inhibitor. No significant relationships were found for other genes unless patients were subgrouped according to CYP2D6 phenotypes or ABCB1 genotypes. Lower concentrations of endoxifen and endoxifen/4‐hydroxytamoxifen ratios were seen with impaired CYP2C9 (P = .05 and P = .03, respectively) if patients had the same CYP2D6 phenotype and were not taking a CYP2D6 or CYP2C19 inhibitor. Lower concentrations of 4‐hydroxytamoxifen were seen for impaired CYP2C19 when ABCB1 SNP3435 was nonvariant (P = .04). With 3 impaired CYP phenotypes, endoxifen concentrations were lower than if only CYP2D6 was impaired (P = .05). When CYP2D6 was impaired, ABCB1 3435 CC (rs1045642) was associated with significantly higher endoxifen (P = .03). Thus, impairment in CYP2C9, CYP2C19, or ABCB1 contributes to a lower steady‐state endoxifen concentration at the dose studied. These studies represent an improved way of examining relationships between pharmacogenetics, drug concentrations, and clinical outcomes and warrants study in larger populations.

Long-term Cross-validation of Everolimus Therapeutic Drug Monitoring Assays: The Zortracker Study

Background: This ongoing academic collaboration was initiated for providing support to set up, validate, and maintain everolimus therapeutic drug monitoring assays and to study long-term interlaboratory performance. Methods: This study was based on EDTA whole blood samples collected from transplant patients treated with everolimus in a prospective clinical trial. Samples were handled under controlled conditions during collection, storage and were shipped on dry ice to minimize freeze–thaw cycles. For more than 1.5 years, participating laboratories received a set of 3 blinded samples on a monthly basis. Among others, these samples included individual patient samples, patient sample pools to assess long-term performance, and patient samples pools enriched with isolated everolimus metabolites. Results: The results between liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and the everolimus Quantitative Microsphere System (QMS, Thermo Fisher) assay were comparable. The monthly interlaboratory variability (coefficient of variation %) for cross-validation samples ranged from 6.5% to 23.2% (average of 14.8%) for LC-MS/MS and 4.2% to 26.4% (average of 11.1%) for laboratories using the QMS assay. A blinded long-term pool sample was sent to the laboratories for 13 months. The result was 5.31 ± 0.86 ng/mL (range, 2.9–7.8 ng/mL) for the LC-MS/MS and 5.20 ± 0.54 ng/mL (range, 4.0–6.8 ng/mL) for QMS laboratories. Enrichment of patient sample pools with 5–25 ng/mL of purified everolimus metabolites (46-hydroxy everolimus and 39-O-desmethyl everolimus) did not affect the results of either LC-MS/MS or QMS assays. Conclusions: Both LC-MS/MS and QMS assays gave similar results and showed similar performance, albeit with a trend toward higher interlaboratory variability among laboratories using LC-MS/MS than the QMS assay.