Evaluation of the Waters MassTrak LC-MS/MS Assay for Tacrolimus and a Comparison to the Abbott Architect Immunoassay.

BACKGROUND: Tacrolimus (Prograf, Advagraf, and FK-506) is the most commonly prescribed calcineurin inhibitor after kidney and liver transplantation. The use of tacrolimus (in conjunction with other drugs) has successfully contributed to the maintenance of solid organ allografts; however, it also exhibits toxic side effects. Therapeutic drug monitoring of tacrolimus is used as an aid to achieve drug concentrations within a narrow therapeutic window. METHODS: The Waters MassTrak Immunosuppressants assay (LC-MS/MS) for the quantification of tacrolimus in whole blood was evaluated for precision, linearity, lower limit of quantification, matrix effects, and accuracy. A method comparison with the Abbott Architect Tacrolimus immunoassay was also performed. RESULTS: The mean concentration (nanograms per milliliter) and coefficient of variation for low, mid, and high patient pools were 0.6% ± 19.9%, 16.0% ± 5.4%, and 31.2% ± 5.8%, respectively. The MassTrak assay was linear from 0.5 to 30.0 ng/mL. Although the MassTrak and Architect assays correlated well (R = 0.97) for patient samples, the MassTrak assay displayed an average negative bias of 18.5% versus Architect (range of 0.0%-36.7%). Analysis of a certified tacrolimus reference material in human whole blood [European Reference Materials (ERM)-DA110a, LGC Standards] on both platforms failed to completely explain the observed difference for patient samples. CONCLUSIONS: Two widely used assays for therapeutic drug monitoring of tacrolimus are not in agreement with one another. Care should be exercised when interpreting results generated on these 2 assay platforms.

A strategy toward the synthesis of C13-oxidized cembrenolides.

An efficient strategy for the construction of C13-oxidized cembrenolides is reported. Central to this strategy is the installation of the C13 hydroxyl group prior to cembrane macrocyclization (via formation of the C1-C2 bond), allowing access to both C13 alcohol epimers. The orientation of the C13 alcohol was found to influence the cyclization mode of the cembranolide scaffold upon furan oxidation, leading to motifs reminiscent to bipinnatolide F, bielschowskysin, and verrillin.

How the VALID Act could affect patient access to laboratory developed testing for therapeutic drug monitoring.

•LDTs are important to lab medicine as they allow flexibility for laboratories to provide testing that patients need.•Many TDMs are LCMS-based and are considered LDTs.•Some TDMs currently have little to no options for obtaining FDA cleared testing.•The VALID Act may threaten the offering of LDTs in the United States.•The current VALID Act has flaws in its legislations which need to be addressed.

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products.

A 24-Hour Extended Calibration Strategy for Quantitating Tacrolimus Concentrations by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Tacrolimus has a low therapeutic index requiring strict control of whole blood concentrations. Although random access immunoassay platforms exist that rapidly provide quantitative values for tacrolimus, LC-MS/MS may provide more accurate quantitation. However, batch testing in many LC-MS/MS assays is not efficient, particularly when testing patients suspected of having tacrolimus toxicity. Extending calibration curve stability beyond the traditionally accepted single batch may facilitate improved turnaround time and reduce testing costs. A 24-h extended calibration of LC-MS/MS tacrolimus was designed and validated to reduce calibrator usage, improve turnaround time, and provide a more efficient workflow for urgent requests. METHODS: Patient samples included in the study were extracted and assayed with coextracted calibrators and quality control in real time. The same patient samples were extracted again 24 h later without coextracted calibrators. The data acquired from the second patient sample extraction was applied to the original calibration curve acquired 24 h prior and compared to the data for the same samples coextracted with calibrators, creating a value set utilizing extended curve stability. RESULTS: A linear regression compared the results using the extended curve to the results of the coextracted acquisitions. This yielded a strong correlation between the 2 data populations, with a slope of 1.0061 and a correlation coefficient of >0.95. The average bias between original patient values and patient values 24 h later was 3.4% across all patient samples. CONCLUSIONS: Patient tacrolimus values were comparable when extracted within 24 h of calibration versus values coextracted with calibrators. Demonstrating comparability within 24 h of calibration allows the laboratory to provide rapid turnaround time for urgent samples without the need for an entirely new calibration curve.

Dilution of Urine Followed by Adulteration in an Attempt to Deceive the Laboratory.

Adulteration of samples submitted for toxicological analyses can present unique challenges to non-forensic clinical laboratories. With the number of overdose-related deaths expected to surpass 60,000 in 2018, it is incumbent on all members of the healthcare team to be active participants in curbing opioid dependence and identifying prescription drug misuse and diversion. Recently published guidelines have sought to provide guidance to laboratories overseeing prescription drug-monitoring programs. We present a case of sample adulteration in an attempt to conceal prescription non-compliance. The patient possessed only an active prescription for hydrocodone but on initial antibody-based screening the sample tested positive for benzodiazepines and oxycodone in addition to opiates. Active communication between the pain management clinic and the clinical laboratory alerted staff to conduct a more thorough investigation including sample validity testing, analyses of paired serum specimens by liquid chromatography tandem mass spectrometry. Analyses revealed the patient submitted a dilute urine specimen with a crushed hydrocodone pill inside in an attempt to hide prescription non-compliance. Previous screenings had been consistent with the medication list raising the question of whether this was an isolated incident or the patient had simply been more successful in manipulating specimens in the past. This case highlights the need for good communication among all members of the healthcare team and the widespread implementation of specimen validity testing for any laboratory that receives samples from pain clinics.

A Young Woman With Recurrent Gestational Hypercalcemia and Acute Pancreatitis Caused by CYP24A1 Deficiency.

The CYP24A1 gene encodes a mitochondrial 24-hydroxylase that inactivates 1,25(OH)2 D. Loss-of-function mutations in CYP24A1 cause hypercalcemia, nephrolithiasis and nephrocalcinosis. We describe a woman with CYP24A1 deficiency and recurrent gestational hypercalcemia. Her first pregnancy, at age 20, resulted with the intrauterine demise of twin fetuses. Postpartum, she developed severe hypercalcemia (14 mg/dL), altered mental status, and acute pancreatitis. Her PTH was suppressed (6 pg/mL) and her 1,25(OH)2 D was elevated (165 and 195 pg/mL on postpartum day 1 and 5, respectively). Between one and three months postpartum, her serum calcium decreased from 11.4 to 10.2 mg/dL while her 1,25(OH)2 D level decreased from 83 to 24 pg/mL. Her 24-hour urine calcium was 277 mg. Six months postpartum, she became pregnant again. At 14 weeks, her albumin-corrected calcium level was 10.4 mg/dL and her 1,25(OH)2 D level exceeded 200 pg/mL. To establish the diagnosis of CYP24A1 deficiency, we showed her 24,25(OH)2 D level to be undetectable (<2 ng/mL). Exon sequencing of the CYP24A1 gene revealed a homozygous, 8-nucleotide deletion in exon 8, causing an S334V substitution and premature termination due to a frame shift (c.999_1006del, p.Ser334Valfs*9). To prevent hypercalcemia, she was advised to discontinue prenatal vitamins, avoid sun exposure and calcium-rich foods, and start omeprazole and a calcium binder (250 mg K-Phos-neutral with meals). Despite these measures, both hypercalcemia (11.5 mg/dL) and acute pancreatitis recurred. Labor was induced and a healthy, normocalcemic boy was delivered. In the absence of lactation, maternal hypercalcemia resolved within 2 months. This report shows that CYP24A1-deficient subjects may be normocalcemic at baseline. Hypercalcemia may be unmasked by pregnancy through the routine use of calciferol-containing prenatal vitamins, increased 1-alpha hydroxylation of VitD by the placenta and maternal kidney, and production of PTHrP by the uteroplacental unit. CYP24A1 deficiency should be considered in patients with unexplained vitamin D-mediated hypercalcemia. © 2016 American Society for Bone and Mineral Research.

Evaluation and comparison of postmortem hydrocodone concentrations in peripheral blood, central blood and liver specimens: a minimal potential for redistribution.

Postmortem changes can alter the concentration of drugs in the vascular compartment as compared with concentrations originally present at the time of death. Numerous drugs have been reported to increase due to postmortem redistribution (PMR). The potential for PMR of hydrocodone, a therapeutic opioid analgesic used to manage pain, is of particular interest due to its wide use. Hydrocodone concentrations in 39 peripheral blood, central blood, and liver specimens were compared. Dihydrocodeine (DHC), a commonly encountered hydrocodone metabolite, was present in 61% of the cases with an average concentration that was 29% of the hydrocodone value. Central blood to peripheral blood hydrocodone ratios were well correlated (R(2)=0.965) with an average (±S.D.) of 1.3 (±0.35) and a median of 1.2. The liver to peripheral blood (L/P) hydrocodone ratio was also well correlated (R(2)=0.915) with an average (±S.D.) of 3.4 (±1.7) L/kg and a median of 3.0 L/kg. This low L/P ratio suggests that hydrocodone is unlikely to undergo substantial PMR changes.

Comparative analysis of hospital and forensic laboratory ethanol concentrations: A 15 month investigation of antemortem specimens.

Quantitative serum alcohol concentrations from regional hospitals (from specimens collected at time of hospital admission) were compared to results from whole blood (from specimens collected at the time of hospital admission) concentrations measured at the San Diego County Medical Examiner's Office (SDCMEO). Over a 15 month period (January 2012 to March 2013), the postmortem forensic toxicology laboratory analyzed a total of 2,321 cases. Of these, 280 were hospital cases (antemortem) representing 12% of the overall Medical Examiner toxicology casework. 59 of the 280 hospital cases (or 21%) screened positive for alcohol (ethanol). 39 of these 59 cases were included in the study based on available specimens for quantitative analyses. This investigation indicated that serum hospital ethanol concentrations correlated well (R(2) = 0.942) with ethanol values determined at SDCMEO (generally measured in whole blood). There was an observed negative bias with an average of -14.1%. A paired t-test was applied to the data and it was shown that this observed bias is statistically significant. These differences in ethanol concentrations could result from differences in specimen, analytical techniques, and/or calibration. The potential for specimen contamination is also discussed.

False-positive interferences of common urine drug screen immunoassays: a review.

Urine drug screen (UDS) immunoassays are a quick and inexpensive method for determining the presence of drugs of abuse. Many cross-reactivities exist with other analytes, potentially causing a false-positive result in an initial drug screen. Knowledge of these potential interferents is important in determining a course of action for patient care. We present an inclusive review of analytes causing false-positive interferences with drugs-of-abuse UDS immunoassays, which covers the literature from the year 2000 to present. English language articles were searched via the SciFinder platform with the strings 'false positive [drug] urine' yielding 173 articles. These articles were then carefully analyzed and condensed to 62 that included data on causes of false-positive results. The discussion is separated into six sections by drug class with a corresponding table of cross-reacting compounds for quick reference. False-positive results were described for amphetamines, opiates, benzodiazepines, cannabinoids, tricyclic antidepressants, phencyclidine, lysergic acid diethylamide and barbiturates. These false-positive results support the generally accepted practice that immunoassay positive results are considered presumptive until confirmed by a second independent chemical technique.

Synthesis of a highly functionalized core of verrillin.

An efficient stereoselective synthesis of furanoverrillin (5), a highly functionalized core of verrillin (1), is reported. The synthetic strategy is based on constructing bicyclic lactone 17 prior to the 10-membered ring macrocyclization. The effect of the C4 methyl group on the furan reactivity is also discussed.

Total synthesis of norcembrenolide B and scabrolide D.

An efficient stereoselective synthesis of norcembrenolide B (8) and scabrolide D (9) is reported. The strategy is inspired by biogenetic relationships of related cembrenoids. Central to this approach is the construction of norbipinnatin J which upon selective C2 deoxygenation and C8 oxygenation produces norrubifolide and norcoralloidolide A. A sequence of site-selective oxidations and skeletal reorganizations then yields, in a divergent manner, compounds 8 and 9. The studies allow revision of the proposed structure of scabrolide D (9), which is identical to norcembrenolide C.