Hana Brozmanova

Liquid chromatography-tandem mass spectrometry method for determination of five antidepressants and four atypical antipsychotics and their main metabolites in human serum.

The rapid and simple ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination parent drugs: sertraline, fluoxetine, citalopram, paroxetine, venlafaxine, clozapine, olanzapine, quetiapine, risperidone, and their active and nonactive metabolites N-desmethylsertraline, norfluoxetine, desmethylcitalopram, didemethylcitalopram, N-desmethylvenlafaxine, O-desmethylvenlafaxine, N-desmethylclozapine, N-desmethylolanzapine, 2-hydroxyolanzapine and 9-hydroxyrisperidone in human serum. Precipitation of serum proteins was performed with a precipitation reagent consisting of 0.05% solution of ZnSO(4)·7H(2)O in acetonitrile/methanol (40:60, v/v). Alprenolol was used as an internal standard. Chromatographic separation was carried out on a BEH C18 column using gradient elution mobile phase A (2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and B (2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Electrospray in positive mode was used for ionization. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring. Analysis time was 5 min. Drugs were separated into three groups with low, medium and high levels. Correlation coefficients of calibration curves were in the range 0.995-1.000. Coefficients of variation were 4.2-9.5% for intra-assay and 3.0-11.9% for inter-assay. Recoveries were 87.1-110% for intra-assay and 88.1-108.2% for inter-assay. The method was fully validated and can be successfully applied for routine analyses.

Quantiferon-CMV Test in Prediction of Cytomegalovirus Infection After Kidney Transplantation

Infection with cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunosuppressed patients, including organ and bone marrow transplant recipients. The majority of CMV disease is caused by reactivation of alatent infection rather that by newly acquired virus. Many techniques have been currently available to aid in the diagnostics of CMV disease. In this report we performed a prospective evaluation of Quantiferon-CMV assay (Cellestis) to determine whether the test is predictive of CMV disease. CD8+ T-cell CMV-specific immunity was assessed in a longitudinal cohort of 14 kidney transplant recipients. According to our data, subjects with higher cellular immune response measured with Quantiferon test had a lower risk of manifestation of CMV infection than subjects with lower responses. Despite the small number of patients and large intra- and interindividual variability of the data in the study, we observed the Quantiferon-CMV assay to be a sensitive specific test to detect a virus-specific T-cell response. We propose that this assay in combination with viral DNA load estimates may prove to be useful to stratify patients at risk of CMV disease.

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods

BACKGROUND The aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods. METHODS Blood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA(SP)), RIA non-specific method (Immunotech, Czech Republic) (RIA(NS)), and high performance liquid chromatography (HPLC) method. RESULTS Although values obtained by RIA(SP) correlated well those obtained by HPLC (RIA(SP)=0.995.HPLC+9.68; r(2)=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA(NS) were 2.57 times higher than the values obtained by RIA(SP) (RIA(SP)=0.356RIA(NS); r(2)=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA(NS)/RIA(SP) values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA(NS)-RIA(SP): HPLC(metab)=0.921.(RIA(NS)-RIA(SP))+21.3; (r(2)=0.746, n=448). CONCLUSION The combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

High-performance liquid chromatographic method for therapeutic drug monitoring of cyclosporine A and its two metabolites in renal transplant patients

A novel fast HPLC method was developed for the determination of cyclosporine A (CyA) and its two metabolites M17 (AM1) and M21 (AM4N) in blood. Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane. Chromatography was carried out using a microbore RP-column under isocratic elution with acetonitrile-methanol-water (200:80:140, v/v/v) at 70 degrees C and a detector set at 205 nm. Linearity for all three compounds was tested in the range of 1-1000 ng/ml. Recovery was 97-109%, and a coefficient of variation was 1.6-8.8% depending on the particular compound and its concentration. The method was used for a group of renal transplant patients having an inadequate response to CyA therapy in order to evaluate the possible role of CyA and its metabolites on the occurrence of hypertension and other toxicological events.

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patients

A LC-MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200 microL of whole blood was precipitated with 10 mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5 mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1 x 50 mm, 1.7 microm using gradient LC-MS/MS analysis in positive electrospray mode. Time of analysis was 5 min. Linearity in blood was 5-2000 microg/L for CsA, AM1, and AM9; 2-500 microg/L for AM4N; and 2-500 microg/L for all substances in lymphocytes. Coefficient of variations was 1.8-9.8% and recovery was 92.0-110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested.

Differences between prescribed daily doses and defined daily doses of antiepileptics--therapeutic drug monitoring as a marker of the quality of the treatment.

OBJECTIVE Prescribed daily doses (PDDs) of antiepileptics (N03A ATC group) were recorded for drugs used in monotherapy or in combination therapy in the University Hospital in Ostrava, Czechia. Plasma levels were used as an indicator of the quality of treatment. METHOD Request and reply forms for therapeutic drug monitoring (TDM) were used as a source of PDDs and plasma levels. The study included 1,144 in-patients examined in the period 1993 - 2004. The differences in PDD were tested by Mann-Whitney-U-test. ATC/DDD index 2005 was used. Doses given in mono- and polytherapy were compared. RESULTS Median PDDs in samples within the therapeutic range (in mg) in mono-/polytherapy were as follows (DDDs in parenthesis): carbamazepine 600/800 (1,000), clonazepam 2.0/2.0 (8), phenytoin 300/300 (300), ethosuximide -/1000 (1,250), lamotrigine 250/200 (300), phenobarbital -/200 (100), primidone 500/625 (1,250), topiramate -/300 (300), valproic acid 750/1,000 (1,500). Median PDDs in polytherapy with antiepileptics not analyzed for TDM were: gabapentin 900 (1,800), levetiracetam 1,500 (1,500), vigabatrin 1,500 (2,000). CONCLUSIONS PDDs in monotherapy were similar or slightly lower than in combination therapy with an exception for lamotrigine, NS. The differences were significant in carbamazepine, p < 0.0001, and valproic acid, p < 0.001. Patients with plasma levels within the therapeutic range were usually treated with similar or slightly higher doses than the remainder. In polytherapy the PDDs were similar to DDDs in carbamazepine, ethosuximide, phenytoin, and topiramate in samples within the therapeutic range when difference +/- 20 per cent was considered as acceptable PDD of levetiracetam was also similar to actual DDD. In general plasma levels tended to be below the therapeutic range. The differences between PDD and DDD of antiepileptics have to be taken into account especially when utilization of different drugs is compared.

Vancomycin removal during low-flux and high-flux extended daily hemodialysis in critically ill septic patients

AIMS To determine the extent of vancomycin removal and vancomycin pharmacokinetics in septic patients with AKI using daily hemodialysis with polysulphone high-flux and low-flux membrane. METHODS Five patients received 6 h daily dialysis with low-flux polysulphone membrane, four patients with high-flux polysulphone membrane. Vancomycin was administered over the last hour of dialysis. The maintenance dose was adjusted based on pre-hemodialysis serum concentrations. Patients were followed up for two days. RESULTS Median percentage of vancomycin removal by low-flux membrane dialysis was 17% (8-38%) and by high-flux membrane dialysis was 31% (13-43%). Vancomycin clearance was only moderately higher in high-flux membrane dialysis (median 3.01 L/h, range 2.34-3.5 L/h) compared to low-flux dialysis (median 2.48 L/h, range 0.53-5.68 L/h) in the first day of the study. About two-fold higher vancomycin clearance in high-flux dialysis (median 3.62 L/h, range 1.37-5.07 L/h) was observed on the second day of the study than low-flux dialysis (median 1.74 L/h, range 0.75-30.94 L/h). CONCLUSIONS Both high-flux and low-flux membrane dialysis remove considerable amounts of vancomycin in critically ill septic patients with AKI. Application of vancomycin after each dialysis was required to maintain therapeutic concentrations.

Serum levels of lamotrigine during delivery in mothers and their infants

PURPOSE We followed up lamotrigine transport through the placenta and analyzed maternal and umibilical cord concentrations, its ratio and maternal lamotrigine clearance in monotherapy and in combinations. METHODS Maternal and umbilical cord concentrations were analyzed during delivery in a cohort of 63 women between 2001 and 2009. The request forms for routine therapeutic drug monitoring were used as the data source. Maternal concentrations were used for the estimation of apparent oral clearance and paired infant and maternal concentrations for estimation of the infant (umibilical cord)/maternal serum concentration ratio. RESULTS The lamotrigine infant/maternal serum concentration ratio ranged in monotherapy from 0.40 to 1.38 (median 0.91). The ratio in monotherapy showed a possible distribution to two subgroups. Concomitant administration of valproic acid significantly increased both maternal and infant lamotrigine concentrations and significantly decreased lamotrigine clearance by about 65%. Co-medication with carbamazepine increased lamotrigine clearance non-significantly. Highly significant correlations were found between maternal and umbilical cord lamotrigine concentrations, both in monotherapy and in the lamotrigine+valproic acid combination. Infant concentrations of valproic acid were found to be about 30% higher and infant concentrations of carbamazepine were found to be about 20% lower than maternal concentrations. CONCLUSIONS Our data from the large cohort showed the interindividual variability of umbilical cord/maternal serum concentration ratio in lamotrigine monotherapy caused probably by the different activity of the placental lamotrigine metabolizing enzymes UGT1A4 and 2B7 associated with genetic polymorphism. The potential teratogenic effect of lamotrigine combination with valproic acid could be associated with the higher lamotrigine and valproic acid concentrations in the fetus.

Gentamicin pharmacokinetics during continuous venovenous hemofiltration in critically ill septic patients

Abstract Objective: Current dosing recommendations for administration of gentamicin to septic patients with acute kidney injury (AKI) on continuous venovenous hemofiltration (CVVH) at a filtration rate of 45 ml/kg/h are missing. Aim: To describe gentamicin pharmacokinetics and to find an optimal dosing regimen in patients on CVVH. Methods: Seven adult patients were included. Patients received loading dose of 240 mg followed by application of maintenance dose every 24 hours. Maintenance dose was adjusted according to gentamicin Cmax/MIC ratio and drug levels simulation using a pharmacokinetic programme. Results: Median total clearance (0·59–0·79 ml/min/kg) was similar to patients with normal renal function; median volume of distribution was higher than observed in non-septic patients (about 0·5 l/kg versus 0·25 l/kg). Patients with diuresis required an increase of gentamicin dose to reach Cmax/MIC ratio. Conclusion: Septic patients with AKI on CVVH (45 ml/kg/h) require a loading dose of 240 mg, followed by therapeutic drug monitoring to optimize maintenance dose.

Fast simultaneous LC/MS/MS determination of 10 active compounds in human serum for therapeutic drug monitoring in psychiatric medication

A UPLC/MS/MS method with simple protein precipitation has been validated for the fast simultaneous analysis of agomelatine, asenapine, amisulpride, iloperidone, zotepine, melperone, ziprasidone, vilazodone, aripiprazole and its metabolite dehydro-aripiprazole in human serum. Alprenolol was applied as an internal standard. A BEH C18 (2.1 × 50 mm, 1.7 µm) column provided chromatographic separation of analytes using a binary mobile phase gradient (A, 2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v; B, 2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v). Mass spectrometric detection was performed in the positive electrospray ionization mode and ion suppression owing to matrix effects was evaluated. The validation criteria were determined: linearity, precision, accuracy, recovery, limit of detection, limit of quantification, reproducibility and matrix effect. The concentration range was as follows: 0.25-1000 ng/mL for agomelatine; 0.25-100 ng/mL for asenapine and iloperidone; 2.5-1000 ng/mL for amisulpride, aripiprazole, vilazodone and zotepine; 2.3-924.6 ng/mL for dehydroaripiprazole; 2.2-878.4 ng/mL for melperone; and 2.2-883.5 ng/mL for ziprasidone. Limits of quantitation below a therapeutic reference range were achieved for all analytes. Intra-run precision of 0.4-5.5 %, inter-run precision of 0.6-8.2% and overall recovery of 87.9-114.1% were obtained. The validated method was successfully implemented into routine practice for therapeutic drug monitoring in our hospital.