Susan Mohamed

Simultaneous HPLC–UV analysis of rufinamide, zonisamide, lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepines (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 microL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 microm Hydro-RP, 250 mm x 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2-40 microg/mL for RFN and 2-80 microg/mL for ZNS. The limit of quantitation was 2 microg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).

Simple and validated HPLC-UV analysis of levetiracetam in deproteinized plasma of patients with epilepsy.

We present a simple, fast and validated method for the determination of the new generation antiepileptic drug (AED) levetiracetam (LEV) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma sample (500 microL) pretreatment was based on simple deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was carried out on a Synergi 4-microm Hydro-RP, 150 mm x 4 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set at 205 nm. Calibration curves were linear (mean correlation coefficient=0.999) over a range of 4-80 microg/mL. The quantitation limit was 2 microg/mL and the absolute recovery was >90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting expensive solid phase or time-consuming liquid-liquid extraction and drying steps is cheaper, faster and simpler than mostly published analytical methods for levetiracetam. Applied to a large population of patients with epilepsy this assay proved very practical in our therapeutic drug monitoring setting (TDM).

Liver transplantation for mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal, recessive disease caused by mutations in the gene encoding thymidine phosphorylase, leading to reduced enzymatic activity, toxic nucleoside accumulation, and secondary mitochondrial DNA damage. Thymidine phosphorylase replacement has been achieved by allogeneic hematopoietic stem cell transplantation, a procedure hampered by high mortality. Based on high thymidine phosphorylase expression in the liver, a 25‐year‐old severely affected patient underwent liver transplantation. Serum levels of toxic nucleosides rapidly normalized. At 400 days of follow‐up, the patients clinical conditions are stable. We propose liver transplantation as a new therapy for MNGIE. Ann Neurol 2016;80:448–455

Levetiracetam clinical pharmacokinetics in elderly and very elderly patients with epilepsy.

We aimed to compare apparent steady-state oral clearance (CL/F) of the antiepileptic drug levetiracetam (LEV) in elderly (66-80 years, n=105) and very elderly (81-96 years, n=70) vs nonelderly (30-65 years, n=97) patients with epilepsy. Median weight-normalized CL/F (mLmin(-1)kg(-1)) decreased from 1.23 (nonelderly) to 0.83 (elderly) and 0.59 (very elderly) (p<0.001). LEV CL/F significantly declines with aging, elderly and very elderly patients requiring an about 30% and 50% lower dose, respectively, compared to nonelderly adults to achieve a given LEV plasma concentration.

Validated UHPLC-MS/MS method for the simultaneous determination of pramipexole and ropinirole in plasma of patients with Parkinson's disease.

A simple and validated ultra high pressure liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of the dopaminergic agents pramipexole and ropinirole in plasma of patients with Parkinsons disease. Following liquid-liquid extraction with tert-butyl methyl ether from 250μL plasma, the separation of the analytes was achieved on a Gemini NX3 column using 10mM pH 6.0 ammonium formate and 10mM ammonium formate in methanol as binary gradient mobile phase at a flow rate of 0.3mL/min. The MS/MS ion transitions were 212.10→153.03 for pramipexole, 261.2→114.2 for ropinirole and 256.1→211 for the internal standard (lamotrigine). The lower limit of quantitation (LLOQ) for both analytes was 80pg/mL and the linearity was determined from 80 to 4000pg/mL for pramipexole and from 200 to 10000pg/mL for ropinirole. Mean recoveries were 94% for PRA and 73% for ROP. Both intra- and inter-assay precision and accuracy were ≤20% at LLOQ concentration and ≤15% at other concentrations. The proposed validated method was successfully applied to measure plasma concentrations of pramipexole and ropinirole in a series of patients with Parkinsons disease on chronic treatment. By grouping the two dopaminergic agents in the same assay, the method allows a large series of patient samples to be processed in a single analytical session.

HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100μL) pretreatment was based on simple deproteinization by 1.2M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4μm Hydro-RP, 150×4mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7mL/min. The UV detector was set at 267nm. The chromatographic run lasted 19min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0μg/mL. The limit of quantitation was 0.5μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.

Simple and rapid validated HPLC-fluorescence determination of perampanel in the plasma of patients with epilepsy

We present a simple and fast high-performance liquid chromatography method with fluorescence detection for the determination of the antiepileptic drug perampanel in human plasma. The chromatographic separation was performed on a Kinetex PFP (100 × 2.6 mm, 4.6 µm) column, using a mobile phase of sodium acetate 0.03 M pH 3.7 and acetonitrile (40/60, v/v), at a flow rate of 0.8 mL/min. Total chromatography time for each run was 5 min. Sample preparation (250 µL) involved only one simple precipitation step by acetonitrile spiked with mirtazapine as internal standard. The method was validated over a concentration range of 20–1000 ng/mL and successfully applied to measure perampanel concentrations in plasma samples obtained from patients with epilepsy. This assay combines the high specificity of fluorescence detection with a very simple and fast sample pretreatment and can offer real advantages over existing methods in terms of simplicity and transferability to a therapeutic drug monitoring setting.

Effect of valproic acid on perampanel pharmacokinetics in patients with epilepsy

We prospectively examined the effect of antiepileptic (AED) cotherapy on steady state plasma concentrations of perampanel (PMP) in epileptic patients. We classified AEDs as strong enzyme inducers (carbamazepine, phenobarbital, phenytoin, oxcarbazepine), not strong enzyme inducers/not inhibitors (levetiracetam, lamotrigine, topiramate, rufinamide, lacosamide, zonisamide, clobazam), and enzyme inhibitors (valproic acid [VPA]). The main outcome was the comparison of PMP plasma concentration to weight‐adjusted dose ratio (C/D; [μg/mL]/mg kg−1 d−1) among comedication subgroups. From 79 patients (42 females, 37 males) aged (mean ± standard deviation) 33 ± 13 years (range = 12‐66 years), 114 plasma samples were collected. Twenty‐eight patients (44 samples) were cotreated with enzyme inducers (group A), 21 (27 samples) with not strong enzyme inducers/not inhibitors (group B), 21 (31 samples) with not strong enzyme inducers/not inhibitors + VPA (group C), and 9 (12 samples) with enzyme inducers + VPA (group D). PMP C/D was reduced (−56%, P < .001) in group A (1.79 ± 0.80) versus group B (4.05 ± 2.16) and increased (P < .001) in group C (6.72 ± 4.04) compared with groups A (+275%), B (+66%), and D (2.76 ± 2.00, +143%). Our study documents the unpublished higher PMP C/D in patients cotreated with VPA. These findings have both theoretical relevance, suggesting better characterization of PMP metabolic pathways with ad hoc studies, and clinical usefulness in managing patients on AED polytherapy.

Novel UHPLC‐MS/MS method for the determination of rotigotine in the plasma of patients with Parkinson's disease

A novel ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid-liquid extraction with tert-butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate-5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra- and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinsons disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma.

Simple and validated UHPLC–MS/MS analysis of nimodipine in plasma and cerebrospinal fluid of patients with subarachnoid haemorrhage

We present a simple, fast and validated method for the determination of nimodipine in plasma and cerebrospinal fluid (CSF) of patients with subarachnoid haemorrhage using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma or CSF 250μL aliquots were pretreated with acetonitrile spiked with lacosamide as internal standard. The chromatographic separation was performed on a Fusion (3μm) 50×2.0mm I.D. column with gradient elution of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile at a flow rate of 0.35mL/min. The MS/MS ion transitions were 419.1→343 for nimodipine and 251.1→91 for the internal standard. The linearity was determined from 2.0 to 40.0ng/mL in plasma and 40.0-800.0pg/mL in CSF. The lower limit of quantitation (LLOQ) of nimodipine was 0.4ng/mL in plasma and 40pg/mL in CSF. The mean recovery for nimodipine was ≥75% in plasma and ≥90% in CSF at all three considered concentrations. Intra- and interassay precision and accuracy were ≤15% at all quality control concentrations in plasma and CSF. The method was applied to measure plasma and CSF concentrations of nimodipine in a series of patients with subarachnoid haemorrhage treated with intravenous nimodipine. The present procedure, omitting time-consuming liquid-liquid extraction and drying steps, is faster, simpler and cheaper than published LC-MS/MS analytical methods for nimodipine in plasma and the first validated one for nimodipine in CSF.