Fast and Easy Simultaneous Determination of Riboflavin, Folic Acid, All-Trans-Retinol and α-Tocopherol in Human Serum by LC/MS/MS for Bariatric Patients.

The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100 mm, 1.7 µm) using a gradient elution with an analysis time of 10 min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000 nmol/L, folic acid 2.5-250 nmol/L, α-tocopherol 0.5-100 µmol/L and all-trans-retinol 25-2500 nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.

Therapeutic monitoring of lacosamide, perampanel, and zonisamide during breastfeeding.

OBJECTIVE: To provide additional information on the transport of the new anti-seizure medications lacosamide, perampanel, and zonisamide in breast milk and breastfed infants. METHODS: Between 2013 and 2022, concentrations of anti-seizure medications were measured in six women with epilepsy (each drug in two patients) using high-performance liquid chromatography. Additionally, concentrations were determined after two consecutive pregnancies in women receiving lacosamide and one woman receiving zonisamide. In all cases, anti-seizure medication concentrations were measured in the maternal serum and breast milk, and five cases, in the infant serum. RESULTS: For lacosamide, the ratios of breast milk/maternal serum concentration varied between 0.77 and 0.93, the ratios of infant/maternal serum concentrations were 0.16 and 0.35, and the ratios of infant serum/milk concentrations were 0.21 and 0.38. For perampanel, the ratios of breast milk/maternal serum concentration were 0.01 and 0.10 and the ratio of infant/maternal serum concentration was 0.36. For zonisamide, the ratios of breast milk/maternal serum concentration varied between 0.76 and 1.26, the ratios of infant/maternal serum concentrations between 0.44 and 0.85, and the ratios of infant serum/milk concentrations between 0.55 and 1.05. CONCLUSIONS: Breastfeeding is recommended for women using lacosamide, perampanel, and zonisamide. However, the actual exposure can only be accurately evaluated by determining the serum concentration of anti-seizure medication in breastfed infants.

Therapeutic monitoring of serum concentrations of acyclovir and its metabolite 9-(carboxymethoxymethyl) guanine in routine clinical practice.

OBJECTIVE: To obtain information on the serum concentrations of acyclovir and its metabolite in routine health care with respect to the renal function. METHODS: This prospective study analyzed data from 27 patients receiving acyclovir intravenously between June 2019 and October 2021. Patients were divided into two subgroups according to the estimated glomerular filtration rate. Serum concentrations of acyclovir and its metabolite 9-(carboxymethoxymethyl) guanine were mainly analyzed on day 5 after the initiation of treatment before the morning dose (trough concentration) and 30 min after the end of the infusion (peak concentration). RESULTS: Trough acyclovir concentrations ranged from 0.8 to 7.6 mg/L and peak concentrations from 6.3 to 25.7 mg/L, and trough metabolite concentrations ranged from 0.12 to 2.30 mg/L and peak concentrations from 0.47 to 2.70 mg/L, respectively. The ratio of trough metabolite and acyclovir concentrations ranged from 0.07 to 0.63 and the ratio of peak concentrations from 0.03 to 0.24. Patients in the subgroup with reduced renal function were significantly older, smaller, and of lower body weight and received significantly lower doses of acyclovir. CONCLUSIONS: A 10-fold difference in the weight-adjusted apparent clearance of acyclovir was observed between patients. This wide interindividual variability in acyclovir pharmacokinetics can lead not only to toxicity but also to suboptimal acyclovir concentrations in severe infections. Therefore, therapeutic monitoring of serum concentrations of acyclovir and its metabolite may be important for optimizing pharmacotherapy, especially in patients with severe clinical conditions.

Liquid chromatography-tandem mass spectrometry method for quantification of gentamicin and its individual congeners in serum and comparison results with two immunoanalytical methods (fluorescence polarization immunoassay and chemiluminiscent microparticle immunoassay).

BACKGROUND: Total gentamicin is a sum of five congeners C1, C1a, C2, C2a and minor C2b, which differ from each other in their methylation on the purpurosamine ring. Liquid chromatography with mass detection (LC-MS/MS) and specified calibration material enables the concentration of total gentamicin and its individual congeners to be analysed. METHODS: 50 µL serum was precipitated with acetonitrile in the presence of 0.5 mol/L formic acid. A RP BEH C18 1.7 µm 2.1x50 mm column maintained at 30 °C and tobramicin as the internal standard were used. Mass detection was performed in positive electrospray. The gentamicin results were compared with fluorescence polarization immunoassay (FPIA) and chemiluminiscent microparticle immunoassay (CMIA). Passing-Bablock regression analysis and Bland-Altman analysis were used. RESULTS: Calibration curves for individual gentamicin congeners were linear with correlation coefficients between 0.997 and 0.998. Recovery was 91.6-102.0% and the coefficients of variation 1.4-8.4%. The total gentamicin concentration was compared with immunoassay FPIA (LC-MSgen = 0.9798xPFIAgen) and CMIA (LC MSgen = 0.9835xCMIAgen) both with significant correlation (p < 0.001). CONCLUSION: The LC-MS/MS method is fast and precise and can be applied to routine TDM in patients. Comparing it to immunoassays makes it possible to measure concentration of gentamicin congeners, which may be important in the case of their different pharmacokinetics.

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanide in human serum by ultra-high-performance liquid chromatography-tandem mass spectrometry.

A simple and rapid ultra-high-performance liquid chromatography coupled with mass spectrometry method was developed for acyclovir and its metabolite 9-carboxymethoxymethylguanine in human serum. After precipitation of serum samples with 0.1% formic acid in acetonitrile/methanol (40:60, v/v), components were separated on a Luna Omega C18 column (1.6 µm; 2.1 × 150 mm) at 40°C. Mobile phase A (2 mmol/L ammonium acetate, 0.1% formic acid in 5% acetonitrile, v/v/v) and mobile phase B (2 mmol/L ammonium acetate, 0.1% formic acid in 95% acetonitrile, v/v/v) were used for gradient elution. A linear calibration curve was obtained over the range of 0.05-50 mg/L, and the correlation coefficients were better than 0.999. The limit of quantitation was 0.05 mg/L for both analytes. The intra- and interday accuracy and precision at three concentration levels ranged between 1.6 and 13.3%, and recoveries were achieved with a range between 92.2 and 114.2%. This method was developed and validated for the therapeutic monitoring of acyclovir in patients.

Therapeutic monitoring of amiodarone and desethylamiodarone after surgical ablation of atrial fibrillation-evaluation of the relationship between clinical effect and the serum concentration.

BACKGROUND: Association between clinical effect and serum concentration of amiodarone (AMI) and its active metabolite desethylamidarone (DEA) in patients after surgical ablation (SA) of atrial fibrillation (AF) has not yet been studied. AIMS: We wanted to find a correlation between AMI and DEA serum concentration and maintaining sinus rhythm (SR) after SA of AF. METHODS: Sixty eight patients with AF who had undergone surgical ablation between 2014 and 2017 were included in a single-centre, prospective, observational study. Maintaining of SR was evaluated by standard 12-lead ECG and 24-hour Holter ECG monitoring at months 1, 3, 6 and 12 following surgery. Therapeutic monitoring of AMI and DEA concentrations was done to optimize therapy and adverse effects were followed up. RESULTS: We have noticed a high success rate in maintaining of SR (overall 83%). The median of serum concentration of AMI was 0.81 mg/L (range 0.16-2.35 mg/L) and DEA 0.70 mg/l (range 0.19-2.63 mg/L). No significant differences were found in the serum concentratration of AMI, DEA or DEA/AMI concentratration ratios between patients with SR and persistent supraventricular tachyarrhythmia except on the second outpatient visit. We observed significant correlation between serum concentration of DEA and thyroid-stimulating hormone elevation. CONCLUSION: We confirmed the efficacy of AMI and DEA at the measured serum concentrations. However, analysis of these concentrations alone cannot replace assessment of the clinical response for treatment. Establishment of individual AMI (and DEA) concentrations at which the optimal therapeutic response is achieved seems to be advantageous. Therapeutic monitoring of AMI and DEA is helpful in personalised pharmacotherapy after SA of AF.

Plasma Levels of Long-Acting Injectable Antipsychotics in Outpatient Care: A Retrospective Analysis.

PURPOSE: Antipsychotic efficacy in schizophrenia depends on its availability in the body. Although therapeutic outcomes remain still far from satisfactory, therapeutic drug monitoring is not a common part of clinical practice during treatment with long-acting injectable antipsychotics (LAI AP). The real effectiveness of LAI AP is thus uncertain. PATIENTS AND METHODS: We made a retrospective evaluation of plasma levels of LAI AP. Collection of blood samples was performed just before the drug application and one week later. Forty patients with a stabilized clinical condition and steady-state plasma levels were included. RESULTS: In the observed cohort of patients, flupentixol decanoate (n = 23) was the most often used drug, followed by fluphenazine decanoate (n = 7), haloperidol decanoate (n = 5), paliperidone palmitate (n = 3), and risperidone microspheres (n = 2). Just 5 of 40 patients were treated with a monotherapy. In the period before the application, 60% of the patients did not reach the therapeutic reference range (TRR) and 20% of the patients had an undetectable plasma level. At the time of collection of the second blood samples performed after 7 days, 24% of the patients were under the TRR. CONCLUSION: We have found a surprisingly high incidence of plasma levels under the TRR in patients treated with LAI AP. Notwithstanding individual variability in pharmacokinetics, it seems that LAI AP may be underdosed in usual clinical practice.

What Does Antidepressant Drug level Monitoring Reveal About Outpatient Treatment and Patient Adherence?

INTRODUCTION: The evaluation of plasma levels of antidepressants may improve the treatment outcome. The aim was to verify adherence and adequacy of administered doses of antidepressants among patients hospitalized for inadequate outpatient therapeutic response. METHODS: Selective serotonin reuptake inhibitors or venlafaxine plasma levels were assessed on the first day of hospitalization and after 3 days of controlled administration. The patients were considered adherent if the plasma level on admission was within the interval of the minimum and maximum plasma level on the fourth day, expanded by 30%. The adequacy of antidepressant doses used during the outpatient treatment was assessed by comparing the plasma level on the fourth day with the therapeutic reference range. RESULTS: Out of 83 patients, 52 (62.7%) were adherent. The plasma levels of antidepressants on the fourth day were found to be within the therapeutic reference range in 35 (43.2%) patients. The same number manifested levels below the therapeutic reference range. In 11 (13.6%) patients, the levels were higher than recommended. No significant difference in rate of adherence was found among individual antidepressants. CONCLUSION: The results show that antidepressant nonresponders are frequently under-dosed or nonadherent.

Lethal suicide attempt with a mixed-drug intoxication of metoprolol and propafenone - A first pediatric case report.

INTRODUCTION: The ß1 adrenergic receptor blocker metoprolol is often prescribed together with the antiarrhythmic drug propafenone. Both are metabolized by cytochrome P450 2D6 and propafenone is also an inhibitor of this enzyme. We present a pediatric case showing metoprolol and propafenone intoxication in combination. CASE: A 14-year-old girl was admitted to a local emergency department after ingestion of metoprolol (probably 1g) and propafenone (probably 1.5-3g) in a suicide attempt. She developed cardiogenic shock with cardiac arrest and was fully resuscitated. Veno-arterial femorofemoral extracorporeal membrane oxygenation was started immediately. High serum levels of both drugs were detected approximately 10h after ingestion (2630ng/mL metoprolol and 2500ng/mL propafenone). Other serial samples for the monitoring of the levels of metoprolol and its metabolite alfa-hydroxymetoprolol were obtained between days 2 and 4 after admission. The metoprolol/alfa-hydroxymetoprolol ratio on the 2nd day was 36.1, indicative of a poor metabolizer phenotype. The elimination half-life of metoprolol was prolonged to 13.2h and the clearance decreased by about 70%. The patient condition gradually worsened, brain edema and intracerebral hemorrhage occurred, and on the 6th day, the patient died. CONCLUSION: We document a pediatric case report of death due to a mixed drug overdose of metoprolol and propafenone, along with data regarding serum metoprolol, alfa-hydroxymetoprolol, and propafenone levels.

New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations.

A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50µL serum was precipitated using 20µL 33% trichloroacetic acid and 0.5mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7µm, 2.1×50mm column maintained at 30°C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5±19.1, 150.2±48.4, 471.7±124.7µmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN=0.947×AbbottVAN+0.192 and LC-MSVAN=0.973×AbbottVAN-0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN=0.866×AbbottVAN+2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.

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.

Therapeutic monitoring of psychoactive drugs - antidepressants: a review.

BACKGROUND: Major depression, is one of the most prevalent mental disorders in Europe and the USA. The dramatic rise in pharmacological antidepressants is mainly due to increase in use of selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors and other new generation antidepressants. In clinical practice, optimum individual doses are often guided by trial-and-error. This article reviews the available literature on therapeutic monitoring of antidepressant drugs. METHODS: A search using MEDLINE (english-language reports, 1983 - August 2012) with the key words for antidepressant drugs and therapeutic drug monitoring. RESULTS: There is a need for monitoring antidepressants due to wide interindividual pharmacokinetic variability. At the same drug dose, a more than 20-fold variation in steady state concentration of drug in the body may result: people differ in their ability to absorb, distribute, metabolise and excrete drugs for reasons of concurrent disease, age, gender, smoking and eating habits, concomitant medication and genetics. CONCLUSIONS: Monitoring of antidepressant drugs enables us to individualise drug doses based on rational therapy, minimalise side effects, reduce morbidity and mortality and cut the cost of health care. Phenotyping and genotyping could increase therapeutic drug monitoring furthere.

Therapeutic drug monitoring of atypical antipsychotic drugs.

Schizophrenia is a severe psychiatric disorder often associated with cognitive impairment and affective, mainly depressive, symptoms. Antipsychotic medication is the primary intervention for stabilization of acute psychotic episodes and prevention of recurrences and relapses in patients with schizophrenia. Typical antipsychotics, the older class of antipsychotic agents, are currently used much less frequently than newer atypical antipsychotics. Therapeutic drug monitoring (TDM) of antipsychotic drugs is the specific method of clinical pharmacology, which involves measurement of drug serum concentrations followed by interpretation and good cooperation with the clinician. TDM is a powerful tool that allows tailor-made treatment for the specific needs of individual patients. It can help in monitoring adherence, dose adjustment, minimizing the risk of toxicity and in cost-effectiveness in the treatment of psychiatric disorders. The review provides complex knowledge indispensable to clinical pharmacologists, pharmacists and clinicians for interpretation of TDM results.

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.

The influence of 7-OH methotrexate metabolite on clinical relevance of methotrexate determination.

BACKGROUND: A modified high performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7-OHMTX) and compared to the immunochemical fluorescence polarization immunoassay (FPIA2) determination of methotrexate. METHODS: Methotrexate was determined by HPLC with UV detection at 303 nm after precipitation of proteins with trichloroacetic acid. Fluorescence polarization immunoassays (FPIA2) of methotrexate were performed on the TDx FLx Immunoassay Analyzer. RESULTS: Our data indicate good correlation between methotrexate levels > 1 micromol/L determined by HPLC and FPIA2. (r = 0.94, Spearman correlation coefficient). However, concentrations of methotrexate < 1 micromol/L measured by fluorescence polarization immunoassay were overestimated. CONCLUSIONS: The concentration of MTX < 1 micromol/L are overestimated due to the cross reactivity with metabolites 7-OHMTX and 2,4-diamino-N10-methylpteroic acid (DAMPA). The cross reaction may affect the therapy and lead to relapse in children with acute lymphoblastic leukemia.