Anton Pohanka

Determination of remifentanil in human plasma by liquid chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation.

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC-MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-(13)C(6) was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at -20°C and for 4h at room temperature while 50% was lost after 24h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.

Determination of four immunosuppressive drugs in whole blood using MEPS and LC–MS/MS allowing automated sample work-up and analysis

In treatment with immunosuppressive drugs, monitoring of blood drug concentration is needed. The aim of this work was to explore micro extraction by packed sorbent (MEPS) as a possible on-line sample preparation method in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantification of cyclosporine, everolimus, sirolimus and tacrolimus in whole blood. An automated on-line MEPS system connected with a LC-MS/MS instrument was set up. A C₈ sorbent was used for the MEPS extraction. Subsequent analysis was performed with a gradient LC system. The adduct ions [M+NH₄]⁺ of the analytes were monitored in SRM mode for quantification. Ascomycin and cyclosporine D were used as internal standards. The chromatographic run time 2.5 min and the quantification ranges were 3-1500 ng/mL (r² ≥ 0.999, n=6) for cyclosporine and 0.5-50 ng/mL for everolimus, sirolimus and tacrolimus (r² ≥ 0.998, 0.994 and 0.993, respectively, n=6). Precision and accuracy were documented at three levels. Accuracy results were between 102% and 109% with precision between 2% and 13% and carry over <0.02%. Matrix effects were characterized and found to be below 20%. The quantifications obtained were in agreement with a reference LC-MS/MS method based on protein precipitation, and results obtained from external proficiency test samples compared with the mean of all other LC-mass spectrometry methods showed good agreement. This method provides an accurate, precise and automated procedure that can be applied for therapeutic drug monitoring of immunosuppressive drugs in clinical laboratories equipped with LC-MS/MS.

Studies on the Antibacterial Effects of Statins - In Vitro and In Vivo

Background Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections. Aim The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract. Methods MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert). Findings The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria. Conclusions Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.

Estimation of dabigatran plasma concentrations in the perioperative setting. An ex vivo study using dedicated coagulation assays.

The perioperative management of dabigatran is challenging, and recommendations based on activated partial thromboplastin time (aPTT) and thrombin time (TT) are unsatisfactory. Dedicated coagulation tests have limitations at plasma concentrations < 50 ng/ml. Therefore, a more sensitive test, which is available 24/7, is required. It was the aim of this study to investigate the performance of the Hemoclot Thrombin Inhibitors® LOW (HTI LOW) kit, a diluted thrombin time, and the STA® - ECA II(ECA-II) kit, a chromogenic variant of the ecarin clotting time, that were developed to measure low dabigatran concentrations, compared to reference dabigatran analysis by liquid chromatography tandem mass-spectrometry (LC-MS/MS). This study included 33 plasma samples from patients treated with dabigatran etexilate who had plasma concentrations < 200 ng/ml. HTI LOW and ECA-II were performed along with HTI, aPTT (STA®-C. K.Prest® and SynthasIL®) and TT (STA® - Thrombin). All procedures were performed according to recommendations by the manufacturers. Linear (or curvilinear) correlations and Bland-Altman analyses were calculated. For free dabigatran concentrations < 50 ng/ml, the R² of linear correlations were 0.69, 0.84 and 0.61, with HTI, HTI LOW and ECA-II, respectively. The R² for TT, STA®-C. K.Prest® and SynthasIL® were 0.67, 0.42 and 0.15. For HTI, HTI LOW and ECA-II, Bland-Altman analyses revealed mean differences of -6 ng/ml (95 %CI: -25-14 ng/ml), 1 ng/ml (95 %CI: -18-19 ng/ml) and -1 ng/ml (95 %CI: -25-23 ng/ml), demonstrating that tests dedicated to measuring low concentrations are more accurate than HTI. In conclusion, the use of HTI LOW or ECA-II to assess low plasma dabigatran concentrations is supported by our findings.

On the monitoring of dabigatran treatment in “real life” patients with atrial fibrillation

INTRODUCTION The oral direct thrombin inhibitor dabigatran is increasingly used to prevent thromboembolic stroke in patients with atrial fibrillation (AF). Routine laboratory monitoring is currently not recommended, but measurements of dabigatran and/or its effect are desirable in certain situations. We studied dabigatran exposure and compared different tests for monitoring of dabigatran in a real-life cohort of AF patients. MATERIAL AND METHODS Ninety AF patients (68 ± 9 years, 67% men, mean CHADS2 score 1.5) were treated with dabigatran 150 (n=73) or 110 mg BID (n=17). Trough plasma concentrations of total and free dabigatran by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to indirect measurements by Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as PT-INR and aPTT. RESULTS Total plasma dabigatran varied 20-fold (12-237 ng/mL with 150 mg BID) and correlated well with free dabigatran (r(2)=0.93). There were strong correlations between LC-MS/MS and HTI or ECA (p<0.001) but these assays were less accurate with dabigatran below 50 ng/mL. The aPTT assay was not dependable and PT-INR not useful at all. There were weak correlations between creatinine clearance (Cockcroft-Gault) and LC-MS/MS, HTI and ECA (p<0.001 for all). A high body weight with normal kidney function was associated with low dabigatran levels. CONCLUSIONS HTI and ECA reflect the intensity of dabigatran anticoagulation, but LC-MS/MS is required to quantify low levels or infer absence of dabigatran. Most real life patients with a normal creatinine clearance had low dabigatran levels suggesting a low risk of bleeding but possibly limited protection against stroke.

A disposable sampling device to collect volume-measured DBS directly from a fingerprick onto DBS paper

BACKGROUND DBS samples collected from a fingerprick typically vary in volume and homogeneity and hence make an accurate quantitative analysis of DBS samples difficult. RESULTS We report a prototype which first defines a precise liquid volume and subsequently stores it to a conventional DBS matrix. Liquid volumes of 2.2 µl ± 7.1% (n = 21) for deionized water and 6.1 µl ± 8.8% (n = 15) for whole blood have been successfully metered and stored in DBS paper. CONCLUSION The new method of collecting a defined volume of blood by DBS sampling has the potential to reduce assay bias for the quantitative evaluation of DBS samples while maintaining the simplicity of conventional DBS sampling.

Does the Russell Viper Venom time test provide a rapid estimation of the intensity of oral anticoagulation? A cohort study

BACKGROUND Dilute Russell Viper Venom Time (DRVV-T) might be useful in urgent settings for screening patients on Non-VKA Oral Anticoagulants (NOACs). AIM To compare the accuracy of DRVV-T with gold standard assays for the assessment of pharmacodynamics of dabigatran, rivaroxaban and vitamin K antagonist (VKA) in plasma samples from patients. METHODS Sixty rivaroxaban, 48 dabigatran and 50 VKA samples from patients were included. DRVV-T was performed in all groups using STA®-Staclot®DRVV-Screen and -Confirm. For NOACs, PT and aPTT were performed using different reagents while plasma drug concentrations were measured by liquid mass-spectrometry (LC-MS/MS). For VKA, INR was performed using RecombiPlasTin 2G®. RESULTS For NOACs, correlations between calibrated STA®-Staclot®DRVV-Confirm and LC-MS/MS (rs=0.88 and 0.97 for rivaroxaban and dabigatran, respectively) were higher than the ones obtained with STA®-Staclot®DRVV-Screen (rs=0.87 and 0.91), PT (rs=0.83 to 0.86) or aPTT (rs=0.84 to 0.89). Bland Altman analyses showed that calibrated DRVV-T methods tend to overestimate plasma concentrations of NOACs. ROC curves revealed that cut-off to exclude supra-therapeutic levels at Ctrough (i.e. 200ng/mL) are different for dabigatran and rivaroxaban. Neither STA®-Staclot®DRVV-Screen nor -Confirm correlated sufficiently with the intensity of VKA therapy (rs=0.35 and 0.52). CONCLUSIONS STA®-Staclot®DRVV-Confirm provides a rapid estimation of the intensity of anticoagulation with rivaroxaban or dabigatran without specific calibrators. At Ctrough, thresholds for rivaroxaban and dabigatran can be used to identify supra-therapeutic plasma level. However, this test cannot differentiate the nature of the NOACs. The development of a point-of-care device optimising this method would be of particular interest in emergency situations.

A LC–MS/MS method for therapeutic drug monitoring of carbamazepine, lamotrigine and valproic acid in DBS

BACKGROUND Therapeutic drug monitoring of antiepileptic drugs in children with epilepsy assists for personalized drug therapy but require numerous patient visits for venous blood sampling. DBS is an alternative matrix applicable to home sampling which can save time and reduce stress for this patient group. RESULTS A fast LC-MS/MS method for quantification of carbamazepine, lamotrigine and valproic acid based on DBS sampling was developed. The method showed linearity in therapeutically relevant concentration ranges and compatible with unknown volume sampling and expected hematocrit range of the patient group. CONCLUSION A LC-MS/MS method for the three most commonly used antiepileptic drugs has been fully validated and clinically applied on DBSs from patients at the neuropediatric clinic at Karolinska University Hospital.

Dabigatran Concentration: Variability and Potential Bleeding Prediction In "Real-Life" Patients With Atrial Fibrillation.

Routine laboratory monitoring is currently not recommended in patients receiving dabigatran despite its considerable variation in plasma concentration. However, in certain clinical situations, measurements of the dabigatran effect may be desirable. We aimed to assess the variability of dabigatran trough and peak concentration and explore the potential relationship between dabigatran concentration and adverse events. We included 44 patients with atrial fibrillation who started treatment with dabigatran 150 mg (D150) or 110 mg (D110) twice daily. They contributed 170 trough and peak blood samples that were collected 2–4 and 6–8 weeks after dabigatran initiation. Plasma dabigatran concentration was measured by LC‐MS/MS and indirectly, by selected coagulation tests. D110 patients were older (74 ± 7 versus 68 ± 6 years), had lower creatinine clearance (68 ± 21 versus 92 ± 24 mL/min) and higher CHA2DS2‐VASc score (3.1 ± 1.3 versus 2.3 ± 0.9) compared to D150 patients (all p < 0.05), but both had similar dabigatran concentrations in both trough and peak samples. Dabigatran concentrations varied less in trough than in peak samples (17.0 ± 13.6 versus 26.6 ± 19.2%, p = 0.02). During the 12‐month follow‐up, 4 patients on D150 and 6 on D110 suffered minor bleeding. There was no major bleeding or thromboembolic event. Patients with bleeding had significantly higher average trough dabigatran concentrations (93 ± 36 versus 72 ± 62 μg/L, p = 0.02) than patients without bleeding, while peak dabigatran values had no predictive value. Dabigatran dose selection according to the guidelines resulted in appropriate trough concentrations with acceptable repeatability. High trough concentrations may predispose patients to the risk of minor bleeding.

Posaconazole Concentrations in Human Tissues after Allogeneic Stem Cell Transplantation

ABSTRACT Few data have been published regarding posaconazole tissue concentrations in humans. We analyzed tissue concentrations in biopsy specimens taken at autopsy from seven patients who received posaconazole prophylaxis because of graft-versus-host disease. The results were compared to plasma concentrations collected before death. Tissue concentrations suggestive of an accumulation of posaconazole were found in the heart, lung, liver, and kidney but not in the brain.