Dysregulation of the Lysophosphatidylcholine/Autotaxin/Lysophosphatidic Acid Axis in Acute-on-Chronic Liver Failure Is Associated With Mortality and Systemic Inflammation by Lysophosphatidic Acid-Dependent Monocyte Activation.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation, monocyte dysfunction, and susceptibility to infection. Lysophosphatidylcholines (LPCs) are immune-active lipids whose metabolic regulation and effect on monocyte function in ACLF is open for study. APPROACHES & RESULTS: Three hundred forty-two subjects were recruited and characterized for blood lipid, cytokines, phospholipase (PLA), and autotaxin (ATX) concentration. Peripheral blood mononuclear cells and CD14+ monocytes were cultured with LPC, or its autotaxin (ATX)-derived product, lysophosphatidic acid (LPA), with or without lipopolysaccharide stimulation and assessed for surface marker phenotype, cytokines production, ATX and LPA-receptor expression, and phagocytosis. Hepatic ATX expression was determined by immunohistochemistry. Healthy volunteers and patients with sepsis or acute liver failure served as controls. ACLF serum was depleted in LPCs with up-regulated LPA levels. Patients who died had lower LPC levels than survivors (area under the receiver operating characteristic curve, 0.94; P < 0.001). Patients with high-grade ACLF had the lowest LPC concentrations and these rose over the first 3 days of admission. ATX concentrations were higher in patients with AD and ACLF and correlated with Model for End-Stage Liver Disease, Consortium on Chronic Liver Failure-Sequential Organ Failure Assessment, and LPC/LPA concentrations. Reduction in LPC correlated with higher monocyte Mer-tyrosine-kinase (MerTK) and CD163 expression. Plasma ATX concentrations rose dynamically during ACLF evolution, correlating with IL-6 and TNF-α, and were associated with increased hepatocyte ATX expression. ACLF patients had lower human leukocyte antigen-DR isotype and higher CD163/MerTK monocyte expression than controls; both CD163/MerTK expression levels were reduced in ACLF ex vivo following LPA, but not LPC, treatment. LPA induced up-regulation of proinflammatory cytokines by CD14+ cells without increasing phagocytic capacity. CONCLUSIONS: ATX up-regulation in ACLF promotes LPA production from LPC. LPA suppresses MerTK/CD163 expression and increases monocyte proinflammatory cytokine production. This metabolic pathway could be investigated to therapeutically reprogram monocytes in ACLF.

Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.

BACKGROUND: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma. METHODS: After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%-96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species. RESULTS: There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at -20 °C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C. CONCLUSIONS: A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4 °C, separated within 2 hours of collection and stored at 4 °C before analysis, with long-term storage at -20 °C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.

A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.

BACKGROUND: Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. METHODS: Whole-blood samples (20 µL) were treated with ammonium bicarbonate (20 µL), zinc sulfate (20 µL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 µL). After centrifugation, 20 µL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). RESULTS: Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 µg/L for sirolimus, 1-41 µg/L for everolimus), and a lower limit of quantitation of 1 µg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. CONCLUSIONS: A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

Measurement of posaconazole, itraconazole, and hydroxyitraconazole in plasma/serum by high-performance liquid chromatography with fluorescence detection.

BACKGROUND: Itraconazole and posaconazole are used in the prevention and treatment of invasive fungal infections. However, the oral bioavailability of both compounds varies widely, and dose-serum concentration relationships are poorly defined for these analytes. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of therapeutic drug monitoring. METHODS: Calibrators (n = 7) and internal quality control solutions (n = 3) were prepared in pooled human serum. Sample (100 µL), internal standard solution (25 µL), Tris solution (2 mol/L; pH 10.6), and extraction solvent (methyl tert-butyl ether, 600 µL) were vortex mixed and centrifuged. The solvent layer was removed and evaporated to dryness and the residue reconstituted in water:methanol (1 + 3, 50 µL). A portion (5 µL) of the reconstituted extract was analyzed using a 3-µm Gemini C6 phenyl column with fluorescence detection (excitation 260 nm, emission 350 nm). The method was used to measure itraconazole and hydroxyitraconazole, or posaconazole, in serum samples taken 1-2 hours before the next dose, from patients forming part of a study into management and diagnostic strategies for invasive aspergillosis. RESULTS: Response was linear over the calibration ranges. Accuracy and imprecision were 92-111.4% and 3.2-13.4% (relative standard deviation), respectively. No interferences were noted. There was a good agreement with nominal values of each analyte in an external quality assessment scheme. In patients prescribed either 400 mg/d of itraconazole (n = 46) or 600-800 mg/d of posaconazole (n = 28) only 24% and 7% of samples, respectively, had serum itraconazole or posaconazole concentrations above the target threshold suggested in published guidelines. CONCLUSIONS: A simple, sensitive high-performance liquid chromatographic method has been developed for the analysis of itraconazole, hydroxyitraconazole, and posaconazole in serum/plasma. Few of the samples measured from patients participating in the clinical study attained concentrations of the drug/metabolite in serum that have been recommended for effective antifungal therapy.

LC-MS in analytical toxicology: some practical considerations.

Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the influence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology.

A rapid and simple assay for lamotrigine in serum/plasma by HPLC, and comparison with an immunoassay.

Monitoring serum/plasma concentrations of lamotrigine may be useful under certain circumstances. An HPLC column packed with strong cation-exchange (SCX)-modified microparticulate silica together with a 100% methanol eluent containing an ionic modifier permits direct injection of sample extracts. An HPLC-UV method developed using this principle for the measurement of serum/plasma lamotrigine is simple, sensitive and selective. The analysis time is less than 5 min. Intra- and inter-assay precision and accuracy meet acceptance criteria, and sample stability, and potential interferences from other compounds have been evaluated. There was good agreement with consensus mean results from external quality assessment samples (n = 32). Analysis of patient samples (n = 115) using the HPLC method and the Seradyn QMS® Lamotrigine immunoassay showed that the immunoassay over-estimated lamotrigine by 21% on average.

HPLC of basic drugs using non-aqueous ionic eluents: evaluation of a 3 mum strong cation-exchange material.

HPLC columns packed with 3 microm particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5 microm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 microm column was not only more efficient for a given column length compared with 5 microm materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3 microm SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required.

The antidepressant desipramine requires the ABCB1 (Mdr1)-type p-glycoprotein to upregulate the glucocorticoid receptor in mice.

The mechanisms by which antidepressants regulate the hypothalamic-pituitary-adrenal (HPA) axis are still unknown. The ABCB1-type multiple drug resistance (MDR) p-glycoprotein (PGP) regulates the HPA axis by limiting the access of glucocorticoids to the brain in mice and humans. Previous work in cell cultures has found that antidepressants enhance glucocorticoid receptor (GR) function in vitro by inhibiting MDR PGP, and therefore by increasing the intracellular concentration of glucocorticoids-but this model has never been tested directly in animals. Here, the tricyclic antidepressant, desipramine (20 mg/kg/day, i.p., for seven days), was administered to abcb1ab MDR PGP knockout mice (congenic on the FVB/N background strain) and to FVB/N controls. The hippocampal mRNA expression of GR, mineralocorticoid receptor (MR), MDR (Mdr1a) PGP, and 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were measured, together with plasma corticosterone levels. In FVB/N controls, desipramine induced a significant upregulation of GR mRNA in the CA1 region (+31%; p=0.045); in contrast, in abcb1ab (-/-) mice, desipramine induced a significant downregulation of GR mRNA in the CA1 region (-45%; p=0.004). MR mRNA expression was unaltered. Desipramine decreased corticosterone levels in both FVB/N controls and in abcb1ab (-/-) mice, but in abcb1ab (-/-) mice the effects were smaller. Specifically, in FVB/N controls (but not in abcb1ab (-/-) mice), desipramine reduced corticosterone levels not only compared with saline-treated mice but also compared with the 'physiological' levels of untreated mice (-39%; p=0.05). Finally, desipramine reduced Mdr1a mRNA expression across all hippocampus areas (-9 to -23%), but had no effect on 11beta-HSD1 mRNA expression. These data support the notion that the MDR PGP is one of the molecular targets through which antidepressants regulate the HPA axis.

Status epilepticus following intentional overdose of fluvoxamine: a case report with serum fluvoxamine concentration.

INTRODUCTION: Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) that is used in the management of depression and obsessive compulsive disorders. We report a patient with status epilepticus requiring quadruple anti-convulsant treatment following a fluvoxamine overdose. CASE REPORT: A 25-year-old female presented with drowsiness at 12 hours following deliberate ingestion of 9.6 grams of fluvoxamine. Sixteen hours after ingestion, she developed status epilepticus that required treatment with benzodiazepines (lorazepam and midazolam), thiopentone, phenytoin and phenobarbitone. Her serum fluvoxamine concentration on presentation was 1970 microg/L (therapeutic 160-220 microg/L) and routine toxicological screening was negative for other drugs. She was discharged home after 72 hours with no further episodes of seizures. DISCUSSION: Most patients with fluvoxamine poisoning are either asymptomatic or may develop mild signs of serotonergic toxicity. Although serotonin syndrome and isolated seizures are reported in fluvoxamine poisoning, we report the first patient with confirmed isolated fluvoxamine toxicity who developed status epilepticus.

LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood.

Therapeutic drug monitoring (TDM) of atypical antipsychotics is common, but published methods often specify relatively complex sample preparation and analysis procedures. The aim of this work was to develop and validate a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in small (200 µL) volumes of plasma or serum for TDM purposes. The applicability of the method as developed to haemolysed whole blood and to oral fluid was also investigated. Analytes and internal standards were extracted into butyl acetate:butanol (9+1, v/v) and a portion of the extract analysed by LC-MS/MS (100 mm × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow-rate 0.5 mL/min; positive ion APCI-SRM, two transitions per analyte). Assay calibration (human plasma, oral fluid, and haemolysed whole blood calibration solutions) was performed by plotting the ratio of the peak area of the analyte to that of the appropriate internal standard. Assay validation was as per FDA guidelines. Assay calibration was linear across the concentration ranges studied. Inter- and intra-assay precision and accuracy were within 10% for all analytes in human plasma. Similar results were obtained for oral fluid and haemolysed whole blood, except that aripiprazole and dehydroaripiprazole were within 15% accuracy at low concentration (15 µg/L) in oral fluid, and olanzapine inter-assay precision could not be assessed in these matrices due to day-by-day degradation of this analyte. Recoveries varied between 16% (sulpiride) and 107% (clozapine), and were reproducible as well as comparable between human plasma, human serum, calf serum and haemolysed whole blood. For oral fluid, recoveries were reproducible, but differed slightly from those in plasma suggesting the need for calibration solutions to be prepared in this medium if oral fluid is to be analysed. LLOQs were 1-5 µg/L depending on the analyte. Neither ion suppression/enhancement, nor interference from some known metabolites of the antipsychotics studied has been encountered. The method has also been applied to the analysis of blood samples collected post-mortem after dilution (1+1, 1+3; v/v) in analyte-free calf serum.