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.

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.

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.

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.

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.

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.

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.

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.

Retrospective Analysis of Pediatric and Adult Populations Using an LC-MS/MS Method for Oxcarbazepine/Eslicarbazepine Metabolite.

BACKGROUND: Therapeutic drug monitoring of anti-epileptic drugs is important to manage seizure control in patients with epilepsy. Oxcarbazepine is a second-generation anti-epileptic drug approved for use in pediatric patients, and eslicarbazepine acetate is a newer generation drug used as adjunctive therapy and monotherapy for partial-onset (focal) seizures. While several second and third generation anti-epileptic drugs have broader therapeutic efficacy in patients, these drugs can still have severe side effects and variable interpatient pharmacokinetics. Consequently, there is a need for accurate and sensitive analytical methods to support therapeutic drug monitoring. METHODS: An assay improvement for a LC-MS/MS method was developed for the major metabolite of oxcarbazepine and eslicarbazepine, licarbazepine (MHD), using a 13C-labeled form of the compound as the internal standard. Additionally, retrospective data analysis was used to compare the distribution of results observed in adult vs pediatric patients. RESULTS: Accuracy and linearity across the analytical measuring range of 1 to 60 µg/mL was acceptable. Inter- and intra-run precision was less than 6% at 3 concentrations tested. The limit of detection was determined to be 0.5 µg/mL. Significant interference from hemolysis, icterus, lipemia, or 187 other potential interferences was not detected. CONCLUSIONS: The improved assay for MHD was appropriate for clinical use. Retrospective data analysis showed that pediatric and adult patients had a similar distribution of oxcarbazepine/eslicarbazepine metabolite concentrations in serum.

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.

Demand for Interpretation of a Urine Drug Testing Panel Reflects the Changing Landscape of Clinical Needs; Opportunities for the Laboratory to Provide Added Clinical Value.

BACKGROUND: The direct detection of drugs and metabolites in urine using a targeted panel offers sensitive and specific detection in comparison to the traditional approach to urine drug testing (screen with reflex of samples with positive results to confirmation testing). The purpose of this study was to evaluate changes in clinical demand for the laboratory to provide interpretation of patient adherence and abstinence, based on reconciling laboratory results and individual patient medication information provided by the clinician. The shifts in toxicology testing likely reflect the inherent complexity of the data and associated interpretation. METHODS: Retrospective testing results associated with a targeted urine drug panel and its related interpretation were collected from our laboratory. We examined the associated testing volume and positivity rates of each reported analyte over 5 consecutive years (2015-2019). Requests from clinicians for consultation regarding this test and use of interpretive comments for the most recent year (2019), as well as access to publicly available educational resources over two years (2018-2019) were collected. RESULTS: The changes in test ordering patterns demonstrate shifting of clinical demands for toxicology testing, by increased adoption of a targeted panel for which laboratory-based interpretation is provided. Positivity rates reflect national shifts in controlled substance prescriptions. Several consultative services were accessed by clinicians suggesting interest and need. CONCLUSION: The value of clinical urine drug testing is improved by providing laboratory-based result interpretation and consultative services.

Cannabinol (CBN) Cross-Reacts with Two Urine Immunoassays Designed to Detect Tetrahydrocannabinol (THC) Metabolite.

BACKGROUND: The psychoactive component of cannabis, tetrahydrocannabinol (THC), is one of many cannabinoids present in the plant. Since cannabinoids have extensive structural similarity, it is important to be aware of potential cross-reactivity with immunoassays designed to detect THC metabolite. This is especially important as cannabinoid products are increasingly marketed as legal supplements. The objective of this study was to assess the cross-reactivity of 2 commercial immunoassays designed to detect THC metabolite with 4 cannabinoids: cannabidiol, cannabinol, cannabichromene, and cannabigerol. METHODS: Deidentified residual patient urine samples that tested negative for THC metabolite on initial testing were pooled and fortified with the above compounds to detect cross-reactivity. We next tested a range of CBN concentrations to determine what concentration of CBN was required to trigger a positive immunoassay result. Finally, we tested whether CBN has an additive effect with THC in the immunoassay by adding CBN to 21 samples weakly positive for THC by a mass spectrometry method but negative by the EMIT II Plus immunoassay. RESULTS: Both the EMIT II Plus assay and the Microgenics MultiGent assay demonstrated cross-reactivity with CBN. For the EMIT II Plus assay, about 5-fold more CBN than THC metabolite was required to produce an assay signal equivalent to the cutoff concentration, and CBN displayed an additive effect with THC metabolite. For the Microgenics assay, 20-fold more CBN than THC metabolite was required to cross the cutoff concentration. CONCLUSIONS: These data may help guide the need for confirmatory testing when results of THC metabolite testing by immunoassay are inconsistent with expectations.

Method validation for a multi-element panel in serum by inductively coupled plasma mass spectrometry (ICP-MS).

BACKGROUND: Laboratory testing for trace and toxic elements is important to diagnose metal toxicity and nutritional deficiency. There are several essential elements that are necessary for biological function and non-essential elements that can pose risk from exposure. Both essential and nonessential elements can be toxic if concentrations exceed a certain threshold. METHODS: An aliquot of serum was diluted in a diluent solution, which contained iridium (Ir) as the internal standard, gold (Au), 0.05% Triton X-100, and 1% nitric acid (HNO3). The diluted specimen was aspirated into an inductively coupled plasma-mass spectrometer for quantitative elemental analysis of chromium (Cr), cobalt (Co), copper (Cu), manganese (Mn), nickel (Ni), selenium (Se) and zinc (Zn). The sample was introduced into the instrument spray chamber to form aerosol droplets, then atomized and ionized in argon plasma. The ions exited the plasma, passed through the interface of the instrument, then arrived at the entrance of the collision cell where helium gas was introduced to remove polyatomic interferences by kinetic energy discrimination (KED). After exiting the collision cell, the ions were filtered by a quadrupole mass spectrometer. RESULTS: The analytical measurement range was determined specifically for each element. Imprecision was <20% CV for the lowest limit of quantification for each element and accuracy was within ±15%. CONCLUSIONS: This method was validated for the quantification of seven elements in serum to assess nutritional deficiency and toxicity. The multi-element panel by ICP-MS met the validation criteria for biological monitoring of trace and toxic elements in patient specimens.

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.

Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device.

BACKGROUND: Therapeutic drug monitoring (TDM) for immunosuppressive (ISP) drugs is an important component of organ and tissue transplantation and chemotherapy management. Whole blood is the specimen type for the quantitative analysis of cyclosporine A, everolimus, sirolimus, and tacrolimus. Some alternatives to venous whole blood samples have the potential to reduce blood volume requirements and simplify sample collection and transport. METHODS: The feasibility of ISP drug (cyclosporine A, everolimus, sirolimus, and tacrolimus) monitoring via microsampling device (MitraTM, Neoteryx) was assessed by comparing venous samples collected and extracted using microsampling device to conventional extraction procedure. Analysis was performed by LC-MS/MS. RESULTS: All analytes were found to be linear across the measurement range of 22.7-937.0 ng/mL (18.9-779.1 nmol/L) for cyclosporine A, 2.3-44.2 ng/mL (2.4-46.1 nmol/L) for everolimus, 2.2-47.2 ng/mL (2.4-51.6 nmol/L) for sirolimus, and 2.2-41.3 ng/mL (2.7-51.4 nmol/L) for tacrolimus. Imprecision was evaluated at concentrations within the therapeutic range and was found to be 10.1% and 5.8% for cyclosporine A, 10.0% and 10.0% for everolimus, 15.0% and 11.9% for sirolimus, and 6.8% and 8.5% for tacrolimus. Method comparison (n = 30 for each analyte, using Deming regression) indicated slopes of 1.08, 1.02, 0.90, and 1.15 and intercepts of -12.8 ng/mL (-10.7 nmol/L), 0.8 ng/mL (0.8 nmol/L), 1.5 ng/mL (1.7 nmol/L), and -0.3 ng/mL (-0.3 nmol/L) for cyclosporine A, everolimus, sirolimus, and tacrolimus, respectively. CONCLUSIONS: This feasibility study demonstrates that precision and bias of ≤15% can be achieved for microsampling-based ISP monitoring.

A 6 Second Analytical Method for Quantitation of Tacrolimus in Whole Blood by Use of Laser Diode Thermal Desorption Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring of immunosuppressive drugs is imperative for organ transplant recipients. High-performance LC-MS/MS is considered gold standard; however, immunoassays provide rapid turnaround time. New technology was developed to reduce mass spectrometry analytical run-time. The laser diode thermal desorption source coupled with tandem mass spectrometry (LDTD-MS/MS) eliminates chromatographic separation to increase analytical throughput. METHODS: A rapid, 6 second, LDTD-MS/MS analytical method was developed for the quantification tacrolimus in whole blood. Whole blood samples were lysed, followed by protein precipitation and solid-phase extraction. Extracted samples with desorption solution were spotted onto a LazWell plate then dried and loaded into the LDTD source for analysis with an AB SCIEX 5500 mass spectrometer in positive multiple reaction monitoring mode. The LDTD laser profile ramps from 0% to 65% of full power over 3 s and is held at 65% for 1 s before returning to initial conditions for 2 s. RESULTS: Data presented include tacrolimus by LDTD-MS/MS comparison to LC-MS/MS, sensitivity, imprecision, interference, linearity, and stability. Method comparison between LDTD-MS/MS and a validated in-house LC-MS/MS assay yielded the following: (LDTD-MS/MS) = 1.119 (LC-MS/MS) + 0.23 ng/mL, Sy/x = 1.26, r = 0.9871 (n = 122). The limit of quantification by LDTD-MS/MS for tacrolimus was <0.3 ng/mL and total imprecision was <10%. CONCLUSIONS: Laser diode thermal desorption tandem mass spectrometry technology can provide rapid turnaround time to result for tacrolimus. The analytical time for LDTD-MS/MS was 6 s compared to 135 s by LC-MS/MS, a >95% decrease in analytical time.