Multiplexed quantification of venlafaxine and metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both are metabolized to inactive metabolites via cytochrome P450 enzymes. While previous studies have focused on quantifying VEN and ODV, bioanalytical methods for the simultaneous measurement of all metabolites are needed to fully characterize the pharmacology of VEN and ODV. METHODS: K2EDTA plasma was spiked with VEN, ODV, N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV). Drugs and metabolites were extracted via protein precipitation and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The multiplexed assay was validated in accordance with regulatory recommendations, and evaluated in remnant plasma samples from persons prescribed venlafaxine. RESULTS: The analytical measuring range for venlafaxine and all four metabolites was 5-800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was between 1.9-9.3% for all levels of all analytes. Minor matrix effects were observed, and both recovery efficiency and process efficiency were >96% for all analytes. All other assay validation assessments met acceptance criteria. Drug concentrations measured from remnant plasma specimens obtained from patients with current venlafaxine prescriptions (37.5-450 mg/day) yielded NDDV, NDV, and NODDV metabolite concentrations in 6/21, 14/21, and 20/21 samples, respectively. The ratio of active to inactive analytes ranged from 0.74 to 14.5, with a median of 6.39. CONCLUSIONS: An efficient and accurate LC-MS/MS method was developed and validated for the quantification of VEN, ODV, and all three inactive metabolites in plasma. The assay met all acceptance criteria, and may be used in future studies of the pharmacokinetics of these drugs.

Solriamfetol and m-chlorophenylpiperazine cause false positive amphetamine results on urine drug screening.

Urine drug screening by immunoassay is a common method to quickly identify drug exposures in the emergency setting and to detect unexpected drug exposures in a variety of patient care and occupational health settings. Although they provide rapid results, immunoassays are susceptible to cross-reactivity with other medications and metabolites. Herein we evaluate the performance of the Thermo Scientific DRI Amphetamines immunoassay for reactivity with trazodone, aripiprazole, atomoxetine, solriamfetol and relevant metabolites. Each of these compounds were spiked into drug-free urine across a range of concentrations and assessed for positivity on amphetamine screen. We demonstrate that the Thermo Scientific DRI assay is susceptible to interferences from m-chlorophenylpiperazine (mCPP), the main metabolite of trazodone, and solriamfetol. Characterization of assay-specific interferences in toxicology screening is instrumental for accurate interpretation of toxicology results, evaluation of patients in emergent settings and supporting patient care.

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.

Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry.

BACKGROUND: Exogenous progestins are an effective tool for hormonal contraception and family planning. Progestins may be delivered as oral pills, intramuscular or subcutaneous injections, vaginal rings, or intrauterine devices. Drug concentrations may vary based on the route and duration of delivery. Measurement of synthetic steroids in blood plasma can aid in determination of product adherence, evaluation of drug-drug interactions, and investigation of unintended pregnancies. METHODS: Drug-free K2EDTA plasma was spiked with the synthetic steroids etonogestrel (ETO), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET). Plasma was combined with isotopically labeled internal standards, and drugs were extracted via liquid-liquid extraction. Samples were then subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated in accordance with regulatory recommendations. The assay was evaluated in a cohort of remnant plasma samples in individuals using one of the aforementioned progestins. RESULTS: The analytical measuring range for ETO, MPA, and NET was 20-10,000 pg/mL; the primary linearity for LNG was 20-20,000 pg/mL. The method showed acceptable precision and accuracy for all progestins. Stability was established for 72 h with room temperature storage and through 3 freeze-thaw cycles. All analytes were stable in whole blood incubated at room temperature for 25 h, and at 40°C and 100% humidity for 2 h. Ion suppression was observed for all analytes spiked in plasma; average ion suppression was 31.6%, 66.6%, 32.1% and 41.2% for ETO, LNG, MPA, and NET, respectively. However, internal standards showed comparable ion suppression, and relative matrix effects were minimal. ETO, LNG, MPA, and NET could also be quantified accurately in K3EDTA plasma and serum. Progestins were successfully measured in remnant samples from individuals using hormonal contraceptives. CONCLUSIONS: A multiplexed LC-MS/MS assay for the quantification of ETO, LNG, MPA, and NET has been developed and validated. The assay met acceptable performance characteristics and may be used in downstream studies to evaluate progestin pharmacology.

Evaluation of serum and rapid serum separator collection tubes for therapeutic drug assays.

Appropriate specimen collection and storage is essential to preserve sample integrity and ensure accurate test results. The default collection containers for blood drug concentrations are tubes without gel separators to avoid possible drug adsorption. However, routine chemistry tests are generally collected in gel separator tubes due to their convenient transport and processing; collection of an additional gel-free tube is often required for drug measurements. Citrated whole blood was pooled, spiked with drug, and transferred to three tubes (red, SST gold, RST orange) containing calcium chloride. Blood was allowed to clot, centrifuged and stored at ambient temperature (24 h) or refrigerated (7 days). At defined times, serum drug concentrations were determined (Roche cobas c502). Based on these results, specimen collection requirements were updated to allow serum separator tubes for 17 assays. Of the 21 assays evaluated, 18 displayed acceptable stability in both gel-containing tubes (acetaminophen, amikacin, carbamazepine, digoxin, ethanol, gentamicin, lamotrigine, levetiracetam, lithium, methotrexate, phenobarbital, phenytoin, salicylate, theophylline, tobramycin, valproic acid, vancomycin, voriconazole). Three drugs displayed strong decreases in measured concentrations after storage in one or both gel-containing tubes (total tricyclics, lidocaine, and free phenytoin). Following adoption of gel-containing tubes, 94% of the five most frequently ordered drug monitoring tests in the Emergency Department were collected in serum separator tubes. Evaluation of the stability and accuracy of commonly monitored drugs revealed that the majority were not affected by exposure to gel separator material under conditions similar to outpatient clinic storage, courier transport, and laboratory storage. Expanding the collection requirements for appropriate drugs to include gel separator tubes decreases the number of specimens drawn and the complexity of laboratory workflows.

Improvement of Umbilical Cord Blood Gas Analysis with Implementation of Clot Catchers.

BACKGROUND: Umbilical cord blood gas testing is a key component of objective pre- and perinatal evaluation of fetal acid base status to determine presence of intrapartum asphyxia and risk of neonatal encephalopathy. Heparinized cord blood is more likely to form small clots than other blood sources, which can interfere with, or preclude, sample analysis. Cord blood samples are irreplaceable and cannot be recollected, thereby compromising clinical decision-making when analysis is not possible. We evaluated processes to prevent excessive rates of cord blood clotting and quantified their impact on successful testing of blood gas specimens. METHODS: Verified result and cancellation data were obtained retrospectively from the laboratory information system. Clot catchers were evaluated using noncord remnant specimens. Collection syringes were compared via collection from remnant cord sections. RESULTS: Prior to implementation of any interventions, retrospective analysis indicated a cancellation rate of 18.6% for umbilical cord blood gas specimens (arterial and venous) and 0.7% for noncord blood arterial and venous samples. Clot catchers were validated for clinical use, with a bias of <±4% for all analytes. After clot catchers were implemented for all cord specimens, cancellation rate decreased approximately 5-fold in the first month and remained <5% a year after implementation. A limited comparison of two heparin syringe types revealed a small difference in the overall rate of specimen clotting. CONCLUSIONS: Implementation of clot catchers was acceptable for analysis of cord blood samples, and when implemented resulted in a sustained 5-fold decrease in the rate of cord blood gas order cancellation.

The non-canonical target PARP16 contributes to polypharmacology of the PARP inhibitor talazoparib and its synergy with WEE1 inhibitors.

PARP inhibitors (PARPis) display single-agent anticancer activity in small cell lung cancer (SCLC) and other neuroendocrine tumors independent of BRCA1/2 mutations. Here, we determine the differential efficacy of multiple clinical PARPis in SCLC cells. Compared with the other PARPis rucaparib, olaparib, and niraparib, talazoparib displays the highest potency across SCLC, including SLFN11-negative cells. Chemical proteomics identifies PARP16 as a unique talazoparib target in addition to PARP1. Silencing PARP16 significantly reduces cell survival, particularly in combination with PARP1 inhibition. Drug combination screening reveals talazoparib synergy with the WEE1/PLK1 inhibitor adavosertib. Global phosphoproteomics identifies disparate effects on cell-cycle and DNA damage signaling thereby illustrating underlying mechanisms of synergy, which is more pronounced for talazoparib than olaparib. Notably, silencing PARP16 further reduces cell survival in combination with olaparib and adavosertib. Together, these data suggest that PARP16 contributes to talazoparib's overall mechanism of action and constitutes an actionable target in SCLC.

Establishing hemolysis and lipemia acceptance thresholds for clinical chemistry tests.

BACKGROUND: A key component of laboratory medicine is the evaluation of specimen suitability for downstream analytical testing. Accurate identification and characterization of the impact of interferents on clinical chemistry analytes is important for patient care. To empirically assess the influence of hemolysis and lipemia on clinical chemistry tests analyzed on a Roche cobas® c701 system, we evaluated serum pools spiked with increasing concentrations of hemolysate and Intralipid®. METHODS: Using an interferent acceptance threshold of within ± 10% of the non-hemolyzed or non-lipemic results, 31 routine chemistry analytes were evaluated. RESULTS: The majority of analytes were determined to have the same or very similar acceptability thresholds as those listed in the vendor package insert. However, several analytes resulted in new thresholds that deviated from manufacturer recommendations (9 higher and 2 lower for lipemia, 7 higher and 6 lower for hemolysis). Samples with high enzyme activities (LDH, ALT, AST, ALP, and CK) were observed to tolerate higher levels of hemolysis, and tiered hemolysis thresholds were established for these enzymes. Independent evaluation of indices is recommended to enable thoughtful implementation of specimen quality criteria and to provide guidance to laboratorians and providers on the nature of these interferences.

Advancements in the gold standard: Measuring steroid sex hormones by mass spectrometry.

Sex hormones, such as testosterone and estrogens, play an essential role in regulating physiological and reproductive development throughout the lifetime of the individual. Although variation in levels of these hormones are observed throughout the distinct stages in life, significant deviations from reference ranges can result in detrimental effects to the individual. Alterations, by either an increase or decrease, in hormone levels are associated with physiological changes, decreased reproductive capabilities, and increased risk for diseases. Hormone therapies (HTs) and assisted reproductive technologies (ARTs) are commonly used to address these factors. In addition to these treatments, gender-affirming therapies, an iteration of HTs, are also a prominent treatment for transgender individuals. Considering that the effectiveness of these treatments relies on achieving therapeutic hormone levels, monitoring of hormones has served as a way of assessing therapeutic efficay. The need for reliable methods to achieve this task has led to great advancements in methods for evaluating hormone concentrations in biological matrices. Although immunoassays are the more widely used method, mass spectrometry (MS)-based methods have proven to be more sensitive, specific, and reliable. Advances in MS technology and its applications for therapeutic hormone monitoring have been significant, hence integration of these methods in the clinical setting is desired. Here, we provide a general overview of HT and ART, and the immunoassay and MS-based methods currently utilized for monitoring sex hormones. Additionally, we highlight recent advances in MS-based methods and discuss future applications and considerations for MS-based hormone assays.

Cancer Chemotherapy: The Case for Therapeutic Drug Monitoring.

The selection of an appropriate therapy and dosing regimen is a significant challenge in the treatment of cancer. Although there are recommended standardized chemotherapy protocols for some types of cancer, protocol changes that usually only occur after large clinical trials demonstrate improvements and individual patients often require dose modifications (amount or interval) or delays in dose administration as toxicities arise. In other areas of medicine, therapeutic drug monitoring is commonly and successfully used to ensure appropriate drug exposure and to limit dose-related toxicities. Currently, the wide pharmacokinetic variability of cytotoxic chemotherapies is addressed clinically by the use of body surface area to determine drug doses; however, this is outdated and demonstrably ineffective for this purpose. This review discusses the challenges of dosing cytotoxic chemotherapies, dose determination strategies for cytotoxic, targeted, and antibody-based biological anticancer drugs, and provides an overview of the recent literature regarding the use of therapeutic drug monitoring in cancer.

Elevated creatinine in a patient on IVIG-therapy.

BACKGROUND: Intravenous immunoglobulin (IVIG) is prepared from the plasma of hundreds of blood donors and is used in the treatment of immunodeficiencies, autoimmune conditions, infections and inflammatory conditions. IVIG formulations contain one of a number of small molecule stabilizers, including sugars, polyols, and amino acids, some of which can cause acute renal failure. CASE REPORT: A 61 y-old woman was treated for paraneoplastic syndrome due to small cell lung cancer in which a creatinine measurement unexpectedly increased during IVIG therapy. This increased creatinine measurement was due to the proline stabilizer in the IVIG fluid that had inadvertently diluted the blood sample. Investigation revealed that proline causes a positive interference in enzymatic creatinine assays, likely due to the activity of sarcosine oxidase, a key component of the assay, on proline. CONCLUSIONS: Increased proline concentrations can occur after administration of high-dose proline (as was the case here) or in the rare metabolic disorders hyperprolinema types I and II.

Clinical Use and Monitoring of Antiepileptic Drugs.

BACKGROUND: Antiepileptic drugs (AEDs) have been used for the treatment of epilepsy and other neurological disorders since the late 19th century. There are currently several classes of AEDs available for epilepsy management, many of which are also used to treat migraines, bipolar disorder, schizophrenia, depression, and neuropathic pain. Because of their molecular and mechanistic diversity, as well as the potential for drug-drug interactions, AEDs are prescribed and monitored in a highly personalized manner. CONTENT: This review provides a general overview of the use of AEDs with a focus on the role of therapeutic drug monitoring. Discussed topics include mechanisms of action, guidelines on the clinical applications of AEDs, clinical tests available for AED monitoring, and genetic factors known to affect AED efficacy. SUMMARY: Implementation of AED therapies is highly individualized, with many patient-specific factors considered for drug and dosage selection. Both therapeutic efficacy and target blood concentrations must be established for each patient to achieve seizure mitigation or cessation. The use of an AED with any additional drug, including other AEDs, requires an evaluation of potential drug-drug interactions. Furthermore, AEDs are commonly used for nonepilepsy indications, often in off-label administration to treat neurological or psychiatric disorders.

Proteome-wide Profiling of Clinical PARP Inhibitors Reveals Compound-Specific Secondary Targets.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a promising class of targeted cancer drugs, but their individual target profiles beyond the PARP family, which could result in differential clinical use or toxicity, are unknown. Using an unbiased, mass spectrometry-based chemical proteomics approach, we generated a comparative proteome-wide target map of the four clinical PARPi, olaparib, veliparib, niraparib, and rucaparib. PARPi as a class displayed high target selectivity. However, in addition to the canonical targets PARP1, PARP2, and several of their binding partners, we also identified hexose-6-phosphate dehydrogenase (H6PD) and deoxycytidine kinase (DCK) as previously unrecognized targets of rucaparib and niraparib, respectively. Subsequent functional validation suggested that inhibition of DCK by niraparib could have detrimental effects when combined with nucleoside analog pro-drugs. H6PD silencing can cause apoptosis and further sensitize cells to PARPi, suggesting that H6PD may be, in addition to its established role in metabolic disorders, a new anticancer target.

Chemoproteomics Reveals Novel Protein and Lipid Kinase Targets of Clinical CDK4/6 Inhibitors in Lung Cancer.

Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably, PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib but not ribociclib.

Synthesis of (±)-actinophyllic acid and analogs: applications of cascade reactions and diverted total synthesis.

Actinophyllic acid is a biologically active indole alkaloid with a unique structural framework that incorporates five contiguous stereocenters. A total synthesis of (±)-actinophyllic acid has been completed that proceeds in only 10 steps from readily available, known compounds and with the isolation of nine intermediates. The synthesis features a novel cascade of reactions of N-stabilized carbocations with π-nucleophiles to create the tetracyclic core of actinophyllic acid in a single chemical operation. This pivotal cascade sequence generates substructures of the actinophyllic acid core that are not otherwise accessible, and one key intermediate was modified to furnish several novel compounds having potentially promising anticancer activity, one of which induces cell death in a wide range of cancer cell lines.

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase.

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1''-2') ribose-ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.

Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase (PARG).

The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling.