Roberta Zilles Hahn

Improved determination of uracil and dihydrouracil in plasma after a loading oral dose of uracil using high-performance liquid chromatography with photodiode array detection and porous graphitic carbon stationary phase

OBJECTIVES The aim of this study was to develop and validate a high-performance liquid chromatographic method for the measurement of plasma concentrations of uracil and dihydrouracil after administration of an oral loading dose of uracil in the context of evaluation of DPD enzyme activity. DESIGN AND METHODS Analytes were extracted from 500μL plasma sampler with a mixture of ethyl acetate isopropanol (85:15, v/v) after protein precipitation with solid ammonium sulfate. The extract was inject in the porous graphitic carbon stationary phase, eluted with water and acetonitrile in gradient mode, allowing complete separation of uracil, dihydrouracil and the internal standard (5-fluorouracil). Chromatograms were monitored at 210 and 260nm. RESULTS Total chromatographic run time, including reequilibration, was 30min. The assay was linear in the concentration range of 0.2 to 20μgmL(-1). Accuracy was 98.4-105.3%, intra-assay precision was 5.1-12.1% and between-assay precision was of 5.3-10.1%. Analytes were stable in plasma at room temperature up to 6h and for three freeze and thaw cycles. Processed samples are stable up to 12h. CONCLUSIONS The developed method was fully validated and has significantly reduced running time when compared to previous assay using porous graphitic stationary phase, allowing complete resolution of uracil, dihydrouracil and internal standard. This assay might be suitable to investigate the eventual correlation between concentrations of uracil and dihydrouracil in plasma after an oral loading dose and DPD enzyme activity, with potential contribution to therapeutic drug monitoring.

Determination of topiramate in dried blood spots using single-quadrupole gas chromatography–mass spectrometry after flash methylation with trimethylanilinium hydroxide

Dried blood spots (DBS) sampling obtained from fingerpricks is a promising and patient friendly alternative for obtaining samples for drug quantification, that could be of interest for topiramate (TOP) therapeutic drug monitoring. The aim of this study was to develop and validate a simple and fast GC-MS assay for TOP measurement in dried blood spots (DBS). The method uses a liquid extraction of one 8mm DBS, followed by a flash methylation with TMAH, and separation in a DB-5ms capillary column. Total analytical run time was 15min. Precision assays presented CV% lower than 9.1% and accuracy was in the range of 94.5-115%. TOP was stable at 25 and 45°C up to 21days. TOP presents saturable binding to red blood cells, resulting in a fraction in plasma (fp) of 0.09-0.03 at 0.8μgml-1 and 0.71-0.45 at 20μgml-1 (both at 25-50 Hct% range). The method was applied to DBS samples obtained after phlebotomy and fingerpicks from an adult individual after oral intake of 100mg TOP (0.25-96h post dose). Plasma and DBS concentrations were moderately correlated (r=0.61), with estimated fp values in the range of 0.06-0.18. Translation of TOP DBS to plasma concentrations is challenging due to its concentration-dependent binding to erythrocytes. Thus, the use of whole blood concentrations for patients monitoring should be considered, which favors to the use of DBS in the clinical context.

Endogenous plasma and salivary uracil to dihydrouracil ratios and DPYD genotyping as predictors of severe fluoropyrimidine toxicity in patients with gastrointestinal malignancies.

OBJECTIVE The aim of this study was to evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratio and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. METHODS Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer, before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *7, *2A, *13 and Y186C. Data on toxicity included grade 1 to 4 neutropenia, mucositis, diarrhea, nausea/vomiting and cutaneous rash. RESULTS 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P<0.01). A cut-off of 1.16 for salivary ratio was set (AUC 0.842), with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 (AUC 0.746), revealed a 71% sensitivity and 76% specificity. CONCLUSIONS DPYD genotyping for alleles 7, *2A, *13 and Y186C was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.

Determinação simultânea de carbamazepina, fenitoína e fenobarbital em sangue seco em papel por cromatografia líquida de alta eficiência

Carbamazepine, phenobarbital and phenytoin were determined in dried blood spots (DBS) by high performance liquid chromatography, after extraction of 8 mm DBS using a mixture of acetonitrile and methanol. Analytes were separated by reversed-phase chromatography, with a run time of 17 minutes. Intra-assay and inter-assay precisions were in the 5.3 to 8.4% and 3.3 to 5.2% ranges, respectively. Accuracy was in the 98.8 to 104.3% range. The method had sensitivity to detect all analytes at levels below minimum therapeutic concentrations. The analytes were stable at 4 oC and room temperature for up to 12 days and at 45 oC for 9 days. The method was applied to 14 paired clinical samples of blood serum and DBS.

Determinação simultânea de topiramato, carbamazepina, fenitoína e fenobarbital em plasma empregando cromatografia a gás com detector de nitrogênio e fósforo

Topiramate and the other frequently co-administered antiepileptic drugs carbamazepine, phenytoin and phenobarbital were determined in 100 µL plasma samples by gas chromatography with nitrogen phosphorus detection (GC-NPD), after a one-step liquid-liquid extraction with ethyl acetate, followed by flash methylation with trimethylphenylammonium hydroxide. Total chromatographic run time was 12.5 min. Intra-assay and inter-assay precision was 2.5-7.3% and 1.6-5.2%, respectively. Accuracy was 100.1-104.2%. The limit of quantitation was 1 µg mL-1 for all analytes, proving suitable for routine application in therapeutic drug monitoring of antiepileptic drugs.

Determination of irinotecan and its metabolite SN-38 in dried blood spots using high-performance liquid-chromatography with fluorescence detection

Irinotecan (IRI) is an antineoplastic drug widely used for the treatment of colorectal and advanced pancreatic cancer. Despite its clinical utility, the clinical use of IRI is associated with potentially severe hematopoietic and gastrointestinal toxicities. The quantification of IRI and its active metabolite SN-38 in dried blood spots (DBS) may be an alternative to individualize the drug dose through a minimally invasive and easy collection method. The aim of this study was to develop and validate a simple and fast HPLC-FL assay for simultaneous IRI and SN-38 measurement in DBS, with adequate analytical performance for clinical use. The method employs liquid extraction of one 8mm disk of whole blood, followed by separation in a reversed phase Eclipse Plus C8 column (150×4.6mm, 5μm). Detection was performed with a fluorescence detector, with excitation wavelength of 370 and emission of 420 for IRI and 540nm for SN-38 and internal standard (camptothecin). Total analytical run time was 17min. Mobile phase was a mixture of 0.1M phosphate buffer pH 4.0 and acetonitrile (80:20, v/v), at 1mLmin-1. The assay was linear in the range 10-3,000ngmL-1 and from 0.5 to 300ngmL-1 for IRI and SN-38, respectively. Precision assays presented CV% of 2.71-5.65 and 2.15-10.07 for IRI and SN-38, respectively, and accuracy in the range of 94.26-100.93 and 94.24-99.33%. IRI and SN-38 were stable at 25 and 42°C for 14days in DBS samples. The method was applied to DBS samples obtained from fingerpicks from 19 volunteers receiving IRI in single or combined chemotherapy regimens, collected 1 and 24h after beginning of the infusion. The estimated plasma concentration of IRI and SN-38 in sample collected 1h after star of infusion had 16 of 19 values within the ±20% range of the measured plasma concentrations. On the other hand, predictions of IRI and SN-38 plasma concentrations from DBS measurements obtained 24h after the beginning of the infusion were poor. AUC of IRI that was calculated using plasma and DBS-estimated concentrations, with a high correlation (r=0.918). The method presented suitable characteristics for the clinical use. However, translation of IRI and SN-38 DBS to plasma concentrations is challenging due to the compounds variable plasma/blood partition.

Analytical and clinical validation of a dried blood spot assay for the determination of paclitaxel using high-performance liquid chromatography-tandem mass spectrometry

BACKGROUND Paclitaxel (PCT) is a chemotherapeutic drug widely used for the treatment of several types of tumors, and its use is associated with severe adverse events, mainly neurologic and hematopoietic toxicities. The relation between systemic exposure and clinical response to PCT was previously described, making paclitaxel a potential candidate for therapeutic drug monitoring (TDM). The use of dried blood spot (DBS) sampling could allow complex sampling schedules required for TDM of PCT. The aim of this study was to develop and validate an LC-MS/MS assay for the quantification of PCT in DBS. METHODS PCT was extracted from one 8 mm DBS punch with a mixture of methanol and acetonitrile, followed by chromatographic separation in a Kinetex C18 (50 × 4.6 mm, 2.6 μm) column. Detection was performed in a 5500-QTRAP® mass spectrometer, with a run time of 2.3 min. RESULTS The assay was linear in the range of 2.5 to 400 ng mL-1. Precision (CV%) and accuracy at the concentration levels of 7.5, 40 and 150 ng mL-1 were 1.69-4.9% and 106.25 to 109.92%, respectively. PCT was stable for 21 days at 25 and 45 °C. The method was applied to DBS samples obtained from 34 patients under PCT chemotherapy. The use of a simple correction factor, derived from the correlation between PCT concentrations in plasma and DBS in this set of patients, allowed unbiased estimation of PCT plasma concentrations from DBS measurements, with similar clinical decisions using either plasma or DBS measurements. CONCLUSIONS DBS testing of PCT concentrations represents a promising alternative for the dissemination of PCT dose individualization.

Simultaneous determination of fluoxetine and norfluoxetine in dried blood spots using high-performance liquid chromatography-tandem mass spectrometry

BACKGROUND Therapeutic drug monitoring (TDM) of the widely prescribed antidepressant fluoxetine (FLU) is recommended in certain situations, such as occurrence of toxicity, inadequate response or suspect of poor adherence. Dried blood spot (DBS) sampling is an increasingly studied alternative for TDM, particularly for outpatients, due to its ease of collection and inherent stability. OBJECTIVES The aim of this study was to develop and validate an LC-MS/MS assay for the simultaneous quantification of FLU and norfluoxetine (NFLU) in DBS. DESIGN AND METHODS The assay is based on a liquid extraction of single DBS with 8mm of diameter, using FLU-D6 as the internal standard, followed by reversed phase separation in an Accucore® C18 column (100×2.1mm, 2.6μm). Mobile phase was composed of water and acetonitrile (gradient from 80:20 to 50:50, v/v), both containing formic acid 0.1%. The assay was validated and applied to 30 patients under FLU pharmacotherapy. RESULTS The assay was linear in the range 10-750ngmL-1. Precision assays presented CV% of 3.13-9.61 and 3.54-7.99 for FLU and NFLU, respectively, and accuracy in the range of 97.98-110.44% and 100.25-105.8%. FLU and NFLU were stable at 25 and 45°C for 7days. The assay was evaluated in 30 patients under FLU treatment. Concentrations of both compounds were higher in DBS than in plasma, and the use of the multiplying factors 0.71 and 0.68 for FLU and NFLU, respectively, allowed acceptable estimation of plasma concentrations, with median prediction bias of -0.55 to 0.55% and mean differences of 0.4 to 2.2ngmL-1. CONCLUSIONS The presented data support the clinical use of DBS for therapeutic drug monitoring of FLU.

Pharmacokinetic and pharmacogenetic markers of irinotecan toxicity

BACKGROUND Irinotecan (IRI) is a widely used chemotherapeutic drug, mostly used for first-line treatment of colorectal and pancreatic cancer. IRI doses are usually established based on patients body surface area, an approach associated with large inter-individual variability in drug exposure and high incidence of severe toxicity. Toxic and therapeutic effects of IRI are also due to its active metabolite SN-38, reported to be up to 100 times more cytotoxic than IRI. SN-38 is detoxified by the formation of SN-38 glucuronide, through UGT1A1. Genetic polymorphisms in the UGT1A1 gene are associated to higher exposures to SN-38 and severe toxicity. Pharmacokinetic models to describe IRI and SN-38 kinetic profiles are available, with few studies exploring pharmacokinetic and pharmacogenetic-based dose individualization. The aim of this manuscript is to review the available evidence supporting pharmacogenetic and pharmacokinetic dose individualization of IRI in order to reduce the occurrence of severe toxicity during cancer treatment. METHODS The PubMed database was searched, considering papers published in the period from 1995-2017, using the keywords irinotecan, pharmacogenetics, metabolic genotyping, dose individualization, therapeutic drug monitoring, pharmacokinetics and pharmacodynamics, either alone or in combination, with original papers being selected based on the presence of relevant data. CONCLUSIONS The findings of this review confirm the importance of considering individual patient characteristics to select IRI doses. Currently, the most straightforward approach for IRI dose individualization is UGT1A1 genotyping. However, this strategy is sub-optimal due to several other genetic and environmental contributions to the variable pharmacokinetics of IRI and its active metabolite. The use of dried blood spot sampling could allow the clinical application of complex sampling for the clinical use of limited sampling and population pharmacokinetic models for IRI doses individualization.

DPD functional tests in plasma, fresh saliva and dried saliva samples as predictors of 5-fluorouracil exposure and occurrence of drug-related severe toxicity

OBJECTIVE to evaluate plasma and salivary uracil (U) to dihydrouracil (UH2) ratios as tools for predicting 5-fluorouracil systemic exposure and drug-related severe toxicity, and clinically validate the use of dried saliva spots (DSS) as an alternative sampling strategy for dihydropyrimidine dehydrogenase (DPD) deficiency assessment. METHODS Pre-chemotherapy plasma, fresh saliva and DSS samples were obtained from gastrointestinal patients (N = 40) for measurement of endogenous U and UH2 concentrations by LC-MS/MS. A second plasma sample collected during 5FU infusion was used for 5FU area under the curve (AUC) determination by HPLC-DAD. Data on toxicity was reported according to CTCAE. RESULTS 15% of the patients developed severe 5FU-related toxicity, with neutropenia accounting for 67% of the cases. U, UH2 and [UH2,]/[U] were highly correlated between fresh and dried saliva samples (rs = 0.960; rs = 0.828; rs = 0.910, respectively). 5FU AUC ranged from 11.3 to 37.31 mg h L-1, with 46.2% of under-dosed and 10.3% over-dosed patients. The [UH2]/[U] ratios in plasma, fresh saliva and dried saliva samples were moderately correlated with 5FU AUC and adverse events grade, indicating a partial contribution of the variables to drug exposure (r = -0.412, rs = -0.373, rs = 0.377) and toxicity (r = -0.363, rs = -0.523, rs = 0.542). Metabolic ratios were lower in patients with severe toxicity (P < .01 salivary ratios, and P < .5 plasma ratios), and 5FU AUC were in average 47% higher in this group than in moderate toxicity. The diagnostic performance of [UH2]/[U] ratios in fresh saliva and DSS for the identification of patients with severe toxicity were comparable. CONCLUSIONS The [UH2]/[U] metabolic ratios in plasma, fresh saliva and DSS were significantly associated with 5FU systemic exposure and toxicity degree. This study also demonstrated the applicability of DSS as alternative sampling for evaluating DPD activity.