One-year retrospective analysis of anti-FXa apixaban and rivaroxaban levels demonstrates utility for management decisions in various urgent and nonurgent clinical situations.

OBJECTIVES: Quantification of direct oral anticoagulant (DOAC) plasma levels can guide clinical management, but insight into clinical scenarios surrounding DOAC-calibrated anti-FXa assays is limited. METHODS: Apixaban- and rivaroxaban-calibrated chromogenic anti-Xa assays performed over a 1-year period were retrospectively analyzed. Patient demographics, DOAC history, concomitant medications, and renal/liver comorbidities were obtained. Indications for testing and associated clinical actions were reviewed. Machine learning (ML) models predicting clinical actions were evaluated. RESULTS: In total, 371 anti-FXa apixaban and 89 anti-FXa rivaroxaban tests were performed for 259 and 67 patients in recurring urgent (acute bleeding, unplanned procedures) and nonurgent situations, including several scenarios not captured by existing testing recommendations (eg, drug monitoring, recurrent thromboembolic events, bleeding tendency). In urgent settings, andexanet reversal was guided by radiologic and clinical findings over DOAC levels in 14 of 32 instances, while 51% of apixaban patients qualified for nonreversal strategies through the availability of levels. Levels also informed procedure/intervention timing and supported management decisions when DOAC clearance or DOAC target levels were in question. The importance of clinical context was emphasized by exploratory ML models predicting particular clinical actions. CONCLUSIONS: Although clinical situations are complex, DOAC testing facilitates clinical decision-making, including reversal, justifying more widespread implementation of these assays.

SERS Detection of the Anti-Epileptic Drug Perampanel in Human Saliva.

Surface-Enhanced Raman Scattering (SERS) can obtain the spectroscopic response of specific analytes. In controlled conditions, it is a powerful quantitative technique. However, often the sample and its SERS spectrum are complex. Pharmaceutical compounds in human biofluids with strong interfering signals from proteins and other biomolecules are a typical example. Among the techniques for drug dosage, SERS was reported to detect low drug concentrations, with analytical capability comparable to that of the assessed High-Performance Liquid Chromatography. Here, for the first time, we report the use of SERS for Therapeutic Drug Monitoring of the Anti-Epileptic Drug Perampanel (PER) in human saliva. We used inert substrates decorated with gold NPs deposited via Pulsed Laser Deposition as SERS sensors. We show that it is possible to detect PER in saliva via SERS after an optimized treatment of the saliva sample. Using a phase separation process, it is possible to extract all the diluted PER in saliva from the saliva phase to a chloroform phase. This allows us to detect PER in the saliva at initial concentrations of the order of 10-7 M, thus approaching those of clinical interest.

Investigation of Fenebrutinib Metabolism and Bioactivation Using MS3 Methodology in Ion Trap LC/MS.

Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3(6H)-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

Islatravir in combination with doravirine for treatment-naive adults with HIV-1 infection receiving initial treatment with islatravir, doravirine, and lamivudine: a phase 2b, randomised, double-blind, dose-ranging trial.

BACKGROUND: Islatravir is a nucleoside reverse transcriptase translocation inhibitor in development for the treatment and prevention of HIV-1 infection. We aimed to assess the efficacy and safety of islatravir-based regimens for the treatment of HIV-1. METHODS: We did a phase 2b, randomised, double-blind, comparator-controlled, dose-ranging trial at 24 clinics or hospitals in four countries (Chile, France, the UK, and the USA). Treatment-naive adults (≥18 years) with plasma HIV-1 RNA concentrations of at least 1000 copies per mL, CD4 T-cell counts of at least 200 cells per mL, and a calculated creatinine clearance of at least 50 mL/min (all within 60 days before study treatment) were eligible for inclusion. Participants were randomly assigned (1:1:1:1) with a block size of four via an interactive voice and web response system to receive oral treatment with one of three doses of islatravir (0·25 mg, 0·75 mg, or 2·25 mg) plus doravirine (100 mg) and lamivudine (300 mg) or to doravirine (100 mg) plus lamivudine (300 mg) plus tenofovir disoproxil fumarate (TDF; 300 mg) once daily with placebo (part 1). Treatment groups were stratified according to screening HIV-1 RNA concentration (≤100 000 copies per mL or >100 000 copies per mL). After at least 24 weeks of treatment, participants taking islatravir who achieved an HIV-1 RNA concentration lower than 50 copies per mL switched to a two-drug regimen of islatravir and doravirine (part 2). All participants and study investigators were masked to treatment in part 1; in part 2, the islatravir dose was masked to all participants and investigators, but the other drugs were given open label. The primary efficacy outcomes were the proportions of participants with an HIV-1 RNA concentration lower than 50 copies per mL at weeks 24 and 48 (US Food and Drug Administration snapshot approach). The primary safety outcomes were the number of participants experiencing adverse events and the number of participants discontinuing study drug owing to adverse events. All participants who received at least one dose of any study drug were included in the analyses. This trial is ongoing, but closed to enrolment of new participants; herein, we report study findings through 48 weeks of treatment. This trial is registered with ClinicalTrials.gov, NCT03272347. FINDINGS: Between Nov 27, 2017, and April 25, 2019, 121 participants (mean age 31 years [SD 10·9], 112 [93%] male, 92 [76%] white, 27 [22%] with HIV-1 RNA concentration >100 000 copies per mL) were randomly assigned: 29 to the 0·25 mg, 30 to the 0·75 mg, and 31 to the 2·25 mg islatravir groups, and 31 to the doravirine, lamivudine, and TDF group. At week 24, 26 (90%) of 29 participants in the 0·25 mg islatravir group, 30 (100%) of 30 in the 0·75 mg islatravir group, and 27 (87%) of 31 in the 2·25 mg islatravir group achieved HIV-1 RNA concentrations lower than 50 copies per mL compared with 27 (87%) of 31 in the doravirine plus lamivudine plus TDF group (difference 2·8%, 95% CI -14·9 to 20·4, for the 0·25 mg islatravir group; 12·9%, -1·6 to 27·5, for the 0·75 mg islatravir group; and 0·3%, -17·9 to 18·5, for the 2·25 mg islatravir group). At week 48, these data were 26 (90%) of 29 in the 0·25 mg islatravir group, 27 (90%) of 30 in the 0·75 mg islatravir group, and 24 (77%) of 31 in the 2·25 mg islatravir group compared with 26 (84%) of 31 in the doravirine plus lamivudine plus TDF group (difference 6·1%, 95% CI -12·4 to 24·4, for the 0·25 mg islatravir group; 6·2%, -12·2 to 24·6, for the 0·75 mg islatravir group; and -6·1%, -27·1 to 14·8, for the 2·75 mg islatravir group). 66 (73%) of participants in the islatravir groups combined and 24 (77%) of those in the doravirine plus lamivudine plus TDF group reported at least one adverse event. Two participants in the 2·25 mg islatravir group and one participant in the doravirine plus lamivudine plus TDF group discontinued owing to an adverse event. No deaths were reported up to week 48. INTERPRETATION: Treatment regimens containing islatravir and doravirine showed antiviral efficacy and were well tolerated regardless of dose. Doravirine in combination with islatravir has the potential to be a potent two-drug regimen that warrants further clinical development. FUNDING: Merck, Sharp, & Dohme Corp, a subsidiary of Merck & Co., Inc.

A simple alternative and improved HPLC method for the estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in solid oral dosage form.

A novel method was developed for the simultaneous estimation of the doravirine, lamivudine, and tenofovir disoproxil fumarate in the pharmaceutical dosage form. The chromatogram was run through an Ascentis C18 column (150 × 4.6 mm, 2.7 µm), with the mobile phase consisting of a phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). The mobile phase was pumped through the column at a flow rate of 1 mL/min. The column temperature was maintained at 30°C. The optimized wavelength for doravirine, lamivudine, and tenofovir disoproxil fumarate was 230.0 nm. The retention times for doravirine, lamivudine, and tenofovir disoproxil were 2.222, 2.764, and 3.403 min, respectively; the relative standard deviation (%) values of method precision for doravirine, lamivudine, and tenofovir disoproxil were 0.6, 0.6, and 0.1, respectively. The % recovery was 100.20%, 100.15%, and 100.36% for doravirine, lamivudine, and tenofovir disoproxil fumarate, respectively. The limit of detection and limit of quantification values were obtained from regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate, and were 0.24 and 0.73 ppm, 0.53 and 1.60 ppm, and 0.47 and 1.43 ppm, respectively. The regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate were y = 17,541x + 117,303, y = 15,555x + 10,791, and y = 15,250x + 31,663, respectively. The method developed was accurate, simple, precise, sensitive, and economical. Hence, it could be adopted for regular quality control for estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in pharmaceutical industries.

Sensing the Anti-Epileptic Drug Perampanel with Paper-Based Spinning SERS Substrates.

The applications of SERS in therapeutic drug monitoring, or other fields of analytical chemistry, require the availability of sensitive sensors and experimental approaches that can be implemented in affordable ways. In this contribution, we show the production of cost-effective SERS sensors obtained by depositing Lee-Meisel Ag colloids on filter paper either by natural sedimentation or centrifugation. We have characterized the morphological and plasmonic features of the sensors by optical microscopy, SEM, and UV-Vis spectroscopy. Such sensors can be used to quantify by SERS the anti-epileptic drug Perampanel (in the concentration range 1 × 10-4-5 × 10-6 M) by spinning them during the micro-Raman measurements on the top of a custom device obtained from spare part hard disk drives. This approach minimizes laser-induced heating effects and allows averaging over the spatial non-uniformity of the sensor.

Synergistic effect of Ficus-zero valent iron supported on adsorbents and Plantago major for chlorpyrifos phytoremediation from water.

Chlorpyrifos and the metabolite 3, 5, 6-trichloro-2-pyridinol (TCP) are widespread contamination of aquatic environments especially freshwater fish. The objectives of this study were to evaluate the contribution of using Ficus zero-valent iron nanoparticles supported on adsorbents (F-Fe0 ad) as green nanotechnology and Plantago major as phytoremediation for removing chlorpyrifos and degradation product TCP polluted water. The shapes of F-Fe0 were circular, with sizes from 2.46 nm to 11.49 nm. Wheat bran (WB) showed the highest extent of removal of chlorpyrifos, while Rice straw ash (RSA) showed the lowest extent of removal. F-Fe0 supported on adsorbents has demonstrated faster removal toward chlorpyrifos compared with tested adsorbents or F-Fe0. Chlorpyrifos was removed more quickly and effectively by P. major L. plus F-Fe0 supported on different adsorbents (nearly 100%) than that by P. major (43.76%) or F-Fe0 (81.69%). The degradation product TCP was more greatly accumulated in water treated with F-Fe0 than that P. major alone or F-Fe0 supported with adsorbents and combined with P. major. Furthermore, TCP significantly accumulated in P. major roots and leaves in the water treated with F-Fe0 supported with adsorbents plus P. major more than that in the P. major roots and leaves alone, this is attributed to the role of F-Fe0 adsorbents for the degradation of chlorpyrifos to TCP, Which strongly accumulated in the P. major roots and leaves. It can be concluded that the contribution of using F-Fe0 supported on adsorbents, especially WB as green nanotechnology and P. major as phytoremediation would be a major role for the complete removal of chlorpyrifos from the water with a significant reduction in the toxic degradation product TCP.

Salting-out assisted liquid-liquid extraction method optimized by design of experiments for the simultaneous high-performance liquid chromatography analysis of perampanel and stiripentol in mouse matrices.

We report a high-performance liquid chromatography method development able to simultaneously determine perampanel and stiripentol, two third-generation antiepileptics whose therapeutic spectrum can potentially be extended, in several mouse matrices. A salting-out assisted liquid-liquid extraction optimized by a design of experiments approach was adopted for sample preparations. Isopropanol and magnesium sulfate were the extraction solvent and salting-out agent, respectively. Both drugs and internal standard (terbinafine) were separated using a LiChroCART® Purospher Star column (C18 , 55 × 4 mm; 3 µm) isocratically pumped with mobile phase [1% triethylamine in water (pH 2.5) and acetonitrile (53:47, v/v)] at 1 mL/min. Stiripentol and terbinafine were detected by fluorescence at 254/372 nm and perampanel at 275/430 nm. Good linearity was demonstrated for perampanel at 1-500 ng/mL range in brain, 2-2000 ng/mL in liver and 1-2000 ng/mL in plasma and kidney (r2  ≥ 0.9922), and for stiripentol between 10 and 2000 ng/mL in brain and 10 and 20 000 ng/mL in the remaining matrices (r2  ≥ 0.9917). Precision (CV ≤ 15%) and accuracy (bias ±15%) were also verified, with obtained recovery values consistent with those predicted by the experimental design. This method was applied in preliminary pharmacokinetic studies to quantify perampanel or stiripentol after oral administration to mice, showing to be a promising bioanalytical tool to support future nonclinical in vivo pharmacokinetic studies.

Fatal intoxication by intravenous injection of castor bean (Ricinus communis L.) extract-a case study.

A case report of a 25-year-old man who committed suicide by intravenous injection himself of an aqueous home-made castor bean extract is presented. The patient was hospitalized and treated symptomatically and was released at its own request fourth day after intoxication. The next day, the patient's condition deteriorated, and he died 6 days after intoxication even though he was given medical care. Case history, autopsy, and toxicological investigation of ante- and post-mortem collected materials are described. Blood and urine collected from the patient ante-mortem and other several biological materials (namely blood from the upper and lower limb, blood from the right and left ventricle, pericardial fluid, vitreous humour, liver, kidney, and spleen) were collected post-mortem during autopsy. Liquid-liquid extraction procedure followed by high-performance liquid chromatography tandem mass spectrometry analysis for identification and determination of ricinine as a biomarker of ricin/castor seed intoxication was developed and validated. The method was applied on analysis of collected ante- and post-mortem biological materials. The post-mortem contents of ricinine in organs (namely the liver, kidney, and spleen) are firstly reported. The obtained results indicated approximately uniform distribution of ricinine (concentration level about 1 ng mL-1) in the body after death. In addition, the GC-MS method was also applied for the analysis of extract of castor seed and the patient's urine, to demonstrate alternative possibility for identification of ricinine for clinical and forensic purposes.

The Effects of Gamma Irradiation on Chemical Biomarker Recovery from Mixed Chemical/Biological Threat Exposure Specimens.

BACKGROUND: Irradiative sterilization of clinical specimens prior to chemical laboratory testing provides a way to not only sterilize pathogens and ensure laboratorian safety but also preserve sample volume and maintain compatibility with quantitative chemical diagnostic protocols. Since the compatibility of clinical biomarkers with gamma irradiation is not well characterized, a subset of diagnostic biomarkers ranging in molecular size, concentration, and clinical matrix was analyzed to determine recovery following gamma irradiation. METHODS: Sample irradiation of previously characterized quality control materials (QCs) at 5 Mrad was carried out at the Gamma Cell Irradiation Facility at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. Following irradiation, the QCs were analyzed alongside non-irradiated QCs to determine analyte recovery between dosed and control samples. RESULTS: Biomarkers for exposure to abrin, ricin, and organophosphorus nerve agents (OPNAs) were analyzed for their stability following gamma irradiation. The diagnostic biomarkers included adducts to butyrylcholinesterase, abrine, and ricinine, respectively, and were recovered at over 90% of their initial concentration. CONCLUSIONS: The results from this pilot study support the implementation of an irradiative sterilization protocol for possible mixed-exposure samples containing both chemical and biological threat agents (mixed CBTs). Furthermore, irradiative sterilization significantly reduces a laboratorian's risk of infection from exposure to an infectious agent without compromising chemical diagnostic testing integrity, particularly for diagnostic assays in which the chemical analyte has been shown to be fully conserved following a 5 Mrad irradiative dose.

Analytical verification and comparison of chromogenic assays for dabigatran, rivaroxaban and apixaban determination on BCSXP and STA Compact Max analysers.

INTRODUCTION: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers. MATERIALS AND METHODS: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis. RESULTS: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x. CONCLUSION: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays.

Development, validation and application of a new HPLC-DAD method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma.

Direct oral anticoagulants (DOACs) have been commonly used for the treatment of venous thromboembolism and for the prevention of stroke in patients with atrial fibrillation. Despite not being initially recommended, monitoring DOACs plasma concentrations is now recognized as essential in emergency situations and in special populations. Moreover, the inter-individual variability found in real studies as well as the high reported non-adherence are corroborating the importance of determining the individual relationship between administered doses, plasma concentrations and pharmacological effects. Therefore, accurate but user-friendly bioanalytical techniques are required to monitor DOACs plasma concentrations in routine clinical practice and phase IV clinical trials. Herein, a fast and simple high performance liquid chromatography (HPLC) method coupled to diode array detection (DAD) was developed, validated and applied to quantify the four currently marketed DOACs (apixaban, edoxaban, dabigatran and rivaroxaban). Sample preparation was performed by solid phase extraction followed by evaporation and concentration of the analytes. Chromatographic separation was accomplished within 6 min on a reversed-phase column (octadecyl-silica packing material; 55 mm × 4 mm, 3 µm particle size), applying a mobile phase composed of an aqueous solution of formic acid (0.1 %, v/v) and acetonitrile, pumped with a gradient elution at 30 °C. The proposed method was linear (r2 ≥ 0.993) within the concentration ranges of 0.017-5.28 µg mL-1, 0.066-5.28 µg mL-1, 0.033-5.28 µg mL-1 and 0.017-5.28 µg mL-1 for apixaban, dabigatran, edoxaban and rivaroxaban, respectively, all of them including the expected range of therapeutic concentrations. Overall, intra- and inter-day trueness of quality control samples, including at the lower limit of quantification (LLOQ), varied between -12.98 to 5.79 %, while imprecision was lower than 16.43 %, supporting that the method is accurate and precise in accordance to international guidelines. Recovery and stability were also assessed and allowed the method to be applied in clinical practice, during therapeutic drug monitoring.

Using the PCI-IS Method to Simultaneously Estimate Blood Volume and Quantify Nonvitamin K Antagonist Oral Anticoagulant Concentrations in Dried Blood Spots.

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy (n = 269) were used to calculate conversion factors. [13C6]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach.

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C18 250 × 4.6 mm column, 5 µm (1.0 ml min-1 , 280 nm, 50 µl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.

Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma.

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe3O4) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe3O4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R2 > 0.9987) over the range (0.001-5.0 µg/mL), limit of detection (0.003 µg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

Biochar amendment effectively reduces the transport of 3,5,6-trichloro-2-pyridinol (a main degradation product of chlorpyrifos) in purple soil: Experimental and modeling.

This study investigates biochar amendment effectively reduces the transport of polar pollutant 3,5,6-trichloro-2-pyridinol, TCP, a main degradation product of chlorpyrifos, and quantitatively explores the physical and chemical mechanisms through inversion simulation. Thus, five biochar addition rates to soil, 0.0%, 0.5%, 1.0%, 2.5% and 5.0%, are tested and compared. The adsorption isotherms experiment, breakthrough curves, BTCs, in both repacked and undisturbed soil columns are also compared. And finally the non-equilibrium convection-diffusion equation, CDE, is used to uncover the change of hydraulic properties of soil and mass non-equilibrium of TCP in the soils mixed with different contents of biochar. The results show that the addition of biochar can reduce the transportation of TCP significantly in the purple soil with macro pores, and the reduction is mainly attributed to two aspects: increase of adsorption ability and decrease of diffusion coefficient and convection velocity. The former is reflected by the linear increase of Kd value with the increase of biochar addition rate and soil organic matter content. The latter is demonstrated by the dramatic reduction of TCP concentration in outflow of BTC experiment and the delayed leaching time. The inversely simulated results also reveal that the diffusion coefficient decrease from 5.35 to 3.95 when biochar addition rate increases from 0 to 5%. Compared with the repacked soil columns, the preferential flow does not disappear in the undisturbed soil columns, accompanied by a higher maximum concentration, an earlier equilibrium time and a less residual amount.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Use of specific anti-Xa levels in acute kidney injury to transition patients from oral factor Xa inhibitors to i.v. heparin infusion.

PURPOSE: This case series presents 3 patients with acute kidney injury taking apixaban or rivaroxaban and transitioning to a heparin infusion. SUMMARY: Case 1 was a 78-year-old man admitted with respiratory failure, acute decompensated heart failure, and acute kidney injury. He was taking apixaban for atrial flutter. He was transitioned to an i.v. heparin infusion and had 2 consecutive heparin antifactor-Xa levels greater than 2 units/mL. Heparin was held and resumed about 36 hours later when the apixaban anti-Xa level was less than 50 ng/mL. Case 2 was a 55-year-old man admitted with acute kidney injury, taking apixaban for a recent deep vein thrombosis. Apixaban anti-Xa levels were monitored and i.v. heparin was initiated when the level was less than 100 ng/mL, about 56 hours after the last apixaban dose. Case 3 was a 64-year-old woman admitted with sepsis and acute kidney injury taking rivaroxaban for pulmonary embolism, which occurred 2 weeks prior to admission. Rivaroxaban anti-Xa levels were monitored and i.v. heparin was initiated about 36 hours after the last dose when the level was less than 100 ng/mL. The management strategy did not lead to any thrombotic outcomes; however, 1 patient experienced bleeding. CONCLUSION: Specific anti-Xa levels for rivaroxaban and apixaban appeared to be helpful in the transition of 3 patients to unfractionated heparin infusions in the setting of acute kidney injury. These levels provided enhanced, individualized care and likely helped avoid over and under anticoagulation.