Biological Testing and Interpretation of Laboratory Results Associated with Detecting Newborns with Substance Exposure.

BACKGROUND: Substance use during pregnancy is common, as is biological testing that is intended to help identify prenatal exposures. However, there is no standardized requirement for biological testing with either maternal or newborn specimens, nor is there standardization related to when testing occurs, how frequently testing occurs, what specimen(s) to test, what substances to test for, or how to perform testing. CONTENT: We review common specimen types tested to detect maternal and newborn substance exposure with a focus on urine, meconium, and umbilical cord tissue. We also review common analytical methods used to perform testing, including immunoassay, and mass spectrometry platforms. Considerations regarding the utilization of testing relative to the purpose of testing, the drug analyte(s) of interest, the specific testing employed, and the interpretation of results are emphasized to help guide decisions about clinical utilization of testing. We also highlight specific examples of unexpected results that can be used to guide interpretation and appropriate next steps. SUMMARY: There are strengths and limitations associated with all approaches to detecting substance exposure in pregnant persons as well as biological testing to evaluate a newborn with possible substance exposure. Standardization is needed to better inform decisions surrounding evaluation of substance exposures in pregnant people and newborns. If biological sampling is pursued, testing options and results must be reviewed in clinical context, acknowledging that false-positive and -negative results can and do occur.

Therapeutic Drug Monitoring and Dosage Adjustments of Immunosuppressive Drugs When Combined With Nirmatrelvir/Ritonavir in Patients With COVID-19.

ABSTRACT: Nirmatrelvir/ritonavir (Paxlovid) consists of a peptidomimetic inhibitor (nirmatrelvir) of the SARS-CoV-2 main protease and a pharmacokinetic enhancer (ritonavir). It is approved for the treatment of mild-to-moderate COVID-19. This combination of nirmatrelvir and ritonavir can mediate significant and complex drug-drug interactions (DDIs), primarily due to the ritonavir component. Indeed, ritonavir inhibits the metabolism of nirmatrelvir through cytochrome P450 3A (CYP3A) leading to higher plasma concentrations and a longer half-life of nirmatrelvir. Coadministration of nirmatrelvir/ritonavir with immunosuppressive drugs (ISDs) is particularly challenging given the major involvement of CYP3A in the metabolism of most of these drugs and their narrow therapeutic ranges. Exposure of ISDs will be drastically increased through the potent ritonavir-mediated inhibition of CYP3A, resulting in an increased risk of adverse drug reactions. Although a decrease in the dosage of ISDs can prevent toxicity, an inappropriate dosage regimen may also result in insufficient exposure and a risk of rejection. Here, we provide some general recommendations for therapeutic drug monitoring of ISDs and dosing recommendations when coadministered with nirmatrelvir/ritonavir. Particularly, tacrolimus should be discontinued, or patients should be given a microdose on day 1, whereas cyclosporine dosage should be reduced to 20% of the initial dosage during the antiviral treatment. Dosages of mammalian target of rapamycin inhibitors (m-TORis) should also be adjusted while dosages of mycophenolic acid and corticosteroids are expected to be less impacted.

Molecular, Viral and Clinical Features of Alcohol- and Non-Alcohol-Induced Liver Injury.

Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.

Impact of the Opioid Epidemic on Drug Testing.

BACKGROUND: This review provides a description of how the opioid epidemic has impacted drug testing. METHODS: Four major service areas of drug testing were considered, including emergency response, routine clinical care, routine forensics, and death investigations. RESULTS: Several factors that the opioid epidemic has impacted in drug testing are discussed, including specimens, breadth of compounds recommended for testing, time to result required for specific applications, analytical approaches, interpretive support requirements, and examples of published practice guidelines. CONCLUSIONS: Both clinical and forensic laboratories have adapted practices and developed new testing approaches to respond to the opioid epidemic. Such changes are likely to continue evolving in parallel with changes in both prescription and nonprescription opioid availability and use patterns, as well as emerging populations that are affected by the "waves" of the opioid epidemic.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Therapeutic Drug Monitoring in Pregnant Patients.

During pregnancy, there are several physiological changes during each trimester that can affect the absorption, distribution, metabolism, and elimination of drugs. Although there is a potential need to understand the pharmacokinetics and pharmacodynamics of drugs in pregnant patients, therapeutic drug monitoring is not well established for various drug classes due to ethical and safety concerns regarding the neonate. Potential risks from in utero drug exposure to the fetus may impact growth and development and may cause malformations or teratogenesis. The clinician must consider the benefits of drug treatment for the pregnant mother versus the risk to the fetus, before prescribing medications during pregnancy. The objective of this review is to aid clinicians, pharmacists, and laboratorians in understanding the pharmacokinetic and pharmacodynamic changes during pregnancy, to provide drug class recommendations for monitoring therapy throughout pregnancy via therapeutic drug monitoring, and to highlight the recent directives of governing agencies on maternal and fetal health.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report.

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.

LC-MS/MS Method for Determination of Teriflunomide, Over a 40,000-Fold Dynamic Range Using Overlapping Calibrators.

BACKGROUND: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been validated for use in therapeutic monitoring of the drug leflunomide in human serum and plasma. Because of concerns of teratogenicity, it is recommended that women who want to become pregnant have concentrations below 0.02 mcg/mL, although therapeutic levels are generally greater than 20 mcg/mL. Consequently, the method required a 40,000-fold dynamic range, which was achieved by dividing the curve range into 2 separate regions but with a single extraction procedure used for both. METHODS: A chromatographic separation was achieved between the parent drug and the active metabolite, teriflunomide (A77 1726), and the latter was quantified across a quantitative range of 0.005-200 mcg/mL. Samples were evaluated in an upper curve region first, with dilution, to determine whether the drug concentrations were in an appropriate therapeutic range. Samples that fell below the upper region were then reevaluated in the lower region without dilution. RESULTS: The method was shown to be reliable, with good accuracy and precision statistics, and acceptable quantitation using 4 different collection tube types. Mean accuracy over 6 control concentrations was within 5.4%, over 5 validation runs, whereas %coefficient of variation (CV) was within 8.15%. Evaluation of sodium heparin, KEDTA, NaF/K oxalate, and plain serum tubes from 6 separate individuals at the lower limit of quantification (LLOQ) showed no influence on the ability to quantify teriflunomide accurately. Regression equations for a curve range of 0.005-1 mcg/mL gave R values of 0.998 or better, whereas the range 0.8-200 mcg/mL had R values of 0.997 or better. CONCLUSIONS: The authors have developed and validated a method that allows quantification of leflunomide across a 40,000-fold range of 0.005-200 mcg/mL.

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.

Retrospective Data Analysis Reveals Unusual Metabolism Pattern of Ethanol in Pediatrics as Compared to Adult and Geriatric Populations.

BACKGROUND: About 95% of consumed ethanol is metabolized by oxidative pathways. Less than 1% is metabolized via nonoxidative pathways: glucuronidation, sulfation, and the formation of fatty acid esters of ethanol. In neonates, the glucuronidation pathway has been reported to be underdeveloped but matures with age. This work compared the test results of patients' random urine samples submitted to our facility for ethyl glucuronide (EtG) and ethyl sulfate (EtS) measurements across pediatric and adult populations. METHODS: Test results (n = 63 498) from urine samples tested for EtG and EtS by quantitative liquid chromatography-tandem mass spectrometry at our facility were utilized for this study. EtG and EtS concentrations were compared across the age partitions 0 to 17 years (pediatric), 18 to 80 years (adult), and 81 to 100 years (geriatric). Eight pediatric patients from a tertiary academic hospital contributed clinical context via abstracted clinical information. RESULTS: Across the individual age partitions, 60% to 65% of patients had both EtG and EtS present in urine. Approximately 5% to 10% of patients had only EtG, and 25% to 35% had neither metabolite present. The lowest percentages (<1.5%) had EtS present in the absence of EtG. Markedly, no pediatric patients had only EtS present; compared to the adult population, this was statistically significant (Fisher exact test, P = 0.025). CONCLUSIONS: From the data presented in this work, EtG is more prevalent relative to EtS in urine samples of patients assessed for ethanol exposure.

A New Broad-Spectrum Drug Screen for 127 Analytes by LC-MS/MS.

BACKGROUND: Broad-spectrum drug screening is offered by many clinical laboratories to support investigation of possible drug exposures. The traditional broad-spectrum drug screen employed at our laboratory utilizes several different analytical platforms, thus requiring relatively high volumes of sample and a cumbersome workflow. Here we describe the development and validation of a consolidated broad-spectrum drug screen assay designed to qualitatively detect 127 compounds in urine (Ur) and serum/plasma (S/P) samples. METHODS: An LC-MS/MS method was developed using the Ultivo LC-MS/MS and designed to be qualitative with a 1-point calibration curve and 50% to 150% controls. Sample preparation included the addition of 122 internal standards (IS) followed by mixed-mode strong cation exchange solid-phase extraction and reverse-phase chromatographic separation on a biphenyl column. RESULTS: For the method described herein, ≥ 95% of analytes in urine and serum control samples had a CV of ≤20% for total imprecision. Accuracy testing included 46 external controls and demonstrated 99.9% accuracy. Method comparison studies to quantitative testing are discussed. The high level of coverage of the analytes with a stable isotope-labeled IS (SIL-IS) helped normalize for matrix effects when significant ion suppression (>25%) was present. Analyte stability in the matrix, the impact of potentially interfering compounds, and method ruggedness were demonstrated. Method limitations include limited detection of glucuronidated drugs and potential cross-contamination with samples at very high concentrations (>>100 × cutoff). CONCLUSIONS: The broad-spectrum drug screen method developed here qualitatively detected 127 drugs and select metabolites. This method could be used to support investigations of possible drug exposures in a clinical setting.

Longitudinal trends in meconium drug detection in 46 US states between the years 2015 and 2020.

Maternal drug use during pregnancy has significant health and socio-legal implications. The Substance Abuse and Mental Health Services Administration publishes self-reported rates of drug use during pregnancy; however, comprehensive long-term laboratory data on neonatal drug exposure are lacking. Over 175,000 meconium specimens originating from 46 US states were analyzed at ARUP Laboratories between the years 2015 and 2020. A retrospective investigation of drug positivity rates, multidrug detection and median drug concentrations was conducted for 28 compounds in six drug classes. The overall meconium drug positivity rate was lowest in 2015 (47.3%), which increased over 6 years, reaching a peak in 2020 (53.4%). 11-Nor-9-carboxy-tetrahydrocannabinol (THC-COOH) was the most frequently detected compound across all 6 years. The second most frequently detected analyte was morphine in 2015-2016 and amphetamines in 2017-2020. The THC-COOH positivity rate rose from 29.7% in 2015 to 38.2% in 2020. The positivity rates for stimulants also increased in the range of 0.4-2.9% in 2020 compared to 2015. Conversely, opioid positivity rates declined in the range of 1.6-2.3% in 2020 as compared to 2015. The most common two-drug combination was THC-COOH-opioids (2.4%) in 2015-2016, which was replaced by THC-COOH-amphetamines (2.6%) in 2017-2020. The most common three-drug combination was THC-COOH-opioids-amphetamines throughout all 6 years. Neonatal drug exposure positivity rates have increased over the past 6 years based on retrospective data analysis from the patient population submitted for testing at ARUP Laboratories.

Trends in Blood Lead Levels Quantified by ICP-MS: A Reference Laboratory Retrospective Study.

BACKGROUND: Exposure to lead may cause severe adverse effects such as anemia, neurologic damage, developmental disorders, and reproductive disorders. Consequently, in 2021, the Centers for Disease Control and Prevention (CDC) reduced its blood lead reference value from 5 µg/dL to 3.5 µg/dL in pediatric patients, 1 to 5 years old. The objective of this study was to perform a retrospective analysis of patient blood lead concentrations reported by ARUP Laboratories to evaluate the frequency of blood lead concentrations greater than 3.5 µg/dL. METHODS: The analysis of blood lead concentration was performed in venous whole blood specimens using inductively coupled plasma mass spectrometry (ICP-MS). In addition, retrospective data analysis was performed to evaluate zinc protoporphyrin (ZPP) concentrations in adult patients with corresponding lead results, using the lead industrial exposure panel. The analysis for ZPP was performed using quantitative hematofluorometry. RESULTS: Retrospective data analysis identified a decline in blood lead concentrations from 2012 to 2021 for pediatric and adult patients. The calculated nonparametric 95% range for ZPP blood was 15 to 43 µg/dL and the ZPP heme ratio 26 to 74 µmol ZPP/mol heme. CONCLUSIONS: Lowering the blood lead reference value (BLRV) to 3.5 µg/dL presents an opportunity for healthcare providers and public health agencies to extend medical or environmental interventions for lead exposure in pediatric patients.

Method validation of multi-element panel in whole blood by inductively coupled plasma mass spectrometry (ICP-MS).

Background: Analytical methods to measure trace and toxic elements are essential to evaluate exposure and nutritional status. A ten-element panel was developed and validated for clinical testing in whole blood. Retrospective data analysis was conducted on patient samples performed at ARUP Laboratories. Methods: A method was developed and validated to quantify ten elements in whole blood by ICP-MS. Fifty microliters of sample were extracted with 950 µL of diluent containing 1 % ammonium hydroxide, 0.1 % Triton X-100, 1.75 % EDTA along with spiked internal standards. Four calibrators were used for each element and prepared in goat blood to match the patient specimen matrix. Samples were analyzed with an Agilent 7700 ICP-MS with a Cetac MVX 7100 µL Workstation autosampler. Results: The assay was linear for all elements with inter- and intra-assay imprecision less than or equal to 11% CV at the low end of the analytical measurement range (AMR) and less than or equal to 4% CV at the upper end of the AMR for all elements. Accuracy was checked with a minimum of 40 repeat patient samples, proficiency testing samples, and matrix-matched spikes. The linear slopes for the ten elements ranged from 0.94 to 1.03 with intercepts below the AMR and R2 ranging from 0.97 to 1.00. Conclusions: The multi-element panel was developed to analyze ten elements in whole blood to unify the sample preparation and increase batch run efficiency. The improved analytical method utilized matrix-matched calibrators for accurate quantification to meet regulatory requirements. The assay was validated according to guidelines for CLIA-certified clinical laboratories and was suitable for clinical testing to assess nutritional status and toxic exposure.

Retrospective analysis of metabolite patterns of clobazam and N-desmethylclobazam in human plasma by LC-MS/MS.

Introduction: Clobazam is a benzodiazepine drug, used to treat Lennox-Gastaut syndrome in patients aged 2 years and older. Objective: To support patient care, our laboratory developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma or serum samples. Methods: The chromatographic separation was achieved with an Agilent Zorbax Eclipse Plus C-18 RRHD column with mobile phase consisting of 0.05% formic acid in 5 mM ammonium formate, pH 3.0 and 0.1% formic acid in acetonitrile at a flow rate of 600 µL/minute and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor precursor-to-product ion transitions in positive electrospray ionization mode. Results: The method was validated over a concentration range of 20-2000 ng/mL for CLB and 200-10,000 ng/mL for N-CLB. The lower limit of quantification was 20 ng/mL for CLB and 200 ng/mL for N-CLB with good accuracy and precision. The method performance was successfully evaluated by comparison with two different external laboratories. Retrospective data analysis was performed to evaluate the positivity rate and metabolic patterns for clobazam from our patient population, as a reference laboratory. Among the positive samples, both parent and metabolite were detected in 96.4% of the samples. Conclusion: The method was developed to support therapeutic drug monitoring and the data generated from retrospective analysis could be useful for result interpretation in conjunction with clinical patient information.

LC-MS/MS Method for Measurement of Thiopurine Nucleotides (TN) in Erythrocytes and Association of TN Concentrations With TPMT Enzyme Activity.

Monitoring concentrations of thiopurine metabolites is used clinically to prevent adverse effects in patients on thiopurine drug therapy. We developed a LC-MS/MS method for the quantification of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in red blood cells (RBCs). This method utilizes an automated cell washer for RBC separation from whole blood samples and washing of the separated RBCs. The lower limit of quantification of the method was 0.2 µmol/L for 6-TG (∼50 pmol/8 × 108 RBC) and 4 µmol/L for 6-MMP (∼1,000 pmol/8 × 108 RBC). The total imprecision of the assay was <3.0%. The upper limit of linearity for 6-TG and 6-MMP was 7.5 µmol/L and 150 µmol/L, respectively. The stability of the thiopurine metabolites under pre- and post-analytically relevant conditions was also evaluated. A good agreement was observed between this method and validated LC-MS/MS methods from three laboratories, except for ∼40% low bias for 6-MMP observed in one of the methods. The assessment of the association between 6-TG and 6-MMP concentrations with thiopurine S-methyltransferase (TPMT) phenotype and genotype demonstrated a statistically significant difference in the thiopurine metabolite concentrations between the TPMT groups with normal and intermediate activity of 6-MMP (p < 0.0001), while the difference in 6-TG concentrations was statistically not significant (p = 0.096). Among the samples with normal TPMT activity, higher concentrations of 6-MMP (p = 0.015) were observed in pediatric samples than in the samples of adults. No statistically significant differences were observed in the distributions of 6-TG and 6-MMP concentrations among the evaluated genotypes.

Tobacco and Cannabis Use During Pregnancy.

OBJECTIVES: Nicotine (NIC) use during pregnancy can influence markers used in biochemical maternal serum screening. This study was designed to determine prevalence of disclosed tobacco smokers in our patient population and to compare disclosed tobacco smoking status with the presence of serum nicotine and a common tetrahydrocannabinol (THC) metabolite. METHODS: A deidentified dataset of disclosed smoking status for quadruple (Quad) screens was obtained. Residual serum submitted for Quad screens was obtained from frozen storage and analyzed for NIC and THC metabolites. RESULTS: Of specimens that had corresponding responses to the smoking history question on the patient history form, 7.2% (n = 1,783 of 24,611) specified that the patient was a tobacco smoker. Of the 271 specimens biochemically analyzed for NIC and THC metabolites, disclosed tobacco smokers had the highest prevalence of detectable NIC and THC metabolites. THC product use was most prevalent in patients categorized as probable tobacco smokers based on cotinine concentrations, as well as in younger patients. CONCLUSIONS: Prevalence and concentration of NIC and THC metabolites vary based on disclosed tobacco smoker status. Biochemical testing may increase sensitivity for the identification of NIC and THC status over self-reporting.

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.

An automated method for supporting busulfan therapeutic drug monitoring.

INTRODUCTION: Busulfan is a chemotherapeutic agent commonly used for myeloablative conditioning regimens such as in the treatment of chronic myelogenous leukemia. Busulfan dosing is complex due to wide interpatient variability in pharmacokinetics and a narrow therapeutic range. Although busulfan dose is normalized to body weight, therapeutic drug monitoring (TDM) using area under the plasma concentration curve is recommended after the first dose. A high busulfan area under the plasma concentration curve (>1500 µM·min) is associated with an increased risk for sinusoidal obstruction syndrome, and a suboptimal area under the plasma concentration curve (<900 µM·min) is associated with an increased risk for graft rejection or disease relapse. TDM of busulfan is not widely available due to the lack of commercially available and rapid methods to determine the area under the plasma concentration curve. METHODS: The purpose of this study was to evaluate the Roche cobas c 111 instrument, a photometric automated chemistry analyzer, using the Busulfan PCM assay from Saladax Biomedical Inc. The assay using this instrument was compared with an enzyme-linked immunosorbent assay (ELISA) from Saladax Biomedical Inc and the Olympus AU400e. Linearity and accuracy were evaluated between 175 and 1750 ng/mL. Imprecision was determined by analyzing 5 concentrations of standards twice a day for 20 days. RESULTS: Linearity for the Roche method had a slope and y-intercept of 1.050 and -5.5, respectively, and percent recovery ranged between 95% and 105%. Correlation between the Roche and ELISA platforms was analyzed by linear regression on 26 frozen patient samples. The results from the comparison of the methods based on the Roche and ELISA platforms were as follows: coefficient of determination (R2) was 0.9684, with a slope and y-intercept of 0.752 and 108.41, respectively. Correlation between the Roche and Olympus instruments was analyzed by linear regression and Bland-Altman plots. The coefficient of determination (R2) was 0.9942, with a slope and y-intercept of 1.035 and -41.3326, respectively. CONCLUSIONS: Availability of TDM of busulfan can be improved by the use of commercially available reagents and automated platforms.