Therapeutic Drug Monitoring of Suramin and Protein Binding

Abstract

A liquid chromatographic method of suramin has been used for the determination of the polysulfonated naphtylurea in both plasma and plasma filtrate from prostate cancer patients treated with the drug. The chromatographic system is based on the use of tetrabutylammonium bromide as an ion-pairing agent, while UV detection at 237 nm and 313 nm is applied. The sample pretreatment is a simple deproteination step by an organic solvent. The same counter-ion as used in the phase system is added in order to increase the recovery of the almost complete protein-bound suramin. The minimum detectable concentration in plasma is ca. 20 ng/mL at 237 nm, in plasma filtrate 10 ng/mL at 237 nm.

The method was routinely applied in plasma level guided treatment of prostate cancer patients with suramin, as well as in protein binding studies. The data of the study demonstrated the necessity of therapeutic drug monitoring in suramin treatment: development of severe, irreversible toxicity could be prevented owing to timed withdrawal of suramin administration when total drug levels were beyond 300 μg/mL. Data of protein binding studies explained in part the development of severe toxicity associated with plasma levels beyond 300–350 μg/mL: at that point free fraction of suramin sharply increases from 500 ng/mL at a total plasma level of 500 μg/mL to 10 μg/mL at a total plasma concentration of 1000 μg/mL, which corresponds with a twenty-fold dose increase (2000%).     

Rapid high-performance liquid chromatographic measurement of buspirone in human plasma after overdose

For toxicological purposes, a rapid reversed-phase high-performance liquid chromatographic method was developed for the determination of the anxiolytic drug, buspirone, in human plasma. A liquid-liquid procedure was used to extract this compound from plasma in the presence of an internal standard, quinupramine. The analysis was performed on a Spherisorb S5 C8 analytical column with UV detection at 240 nm. No endogenous compounds were found to interfere. A linear response was observed over the concentration range 5-100 ng/mL. A good accuracy (bias < or =7.9%) was achieved for all quality controls, with intra-day and inter-day variation coefficients equal or less than 7.6%. The limit of quantification was 5 ng/mL. Stability of buspirone in plasma stored at different temperatures was checked. This rapid method (run time <12 min) was used to manage an acute poisoning involving buspirone.     

Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid–liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C₁₈, 150 mm × 2.0 mm, 5 μm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.     

Pharmacokinetics of dronedarone in rat using a newly developed high‐performance liquid chromatographic assay method

A sensitive and selective high‐performance liquid chromatographic method for the determination of dronedarone in rat plasma was developed. Dronedarone was extracted using one‐step liquid–liquid extraction. The separation of dronedarone was accomplished using a C₁₈ analytical column. The mobile phase was composed of a combination of monobasic potassium phosphate and acetonitrile. The UV detection was at 254 nm for ethopropazine, the internal standard, and after its elution, changed to 290 nm for dronedarone detection. The total analytical run time was 20 min. Mean recovery was >80%; the assay had excellent linear relationships (>0.999) between peak height ratios and plasma concentrations; the lower limit of quantification 25 was ng/mL, based on 100 μL of rat plasma. Accuracy and precision were <18% over the concentration range of 25–500 ng/mL. The assay was applied successfully to the measurement of dronedarone plasma concentrations in rats given the drug orally. Copyright © 2014 John Wiley & Sons, Ltd.     

A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine.

A new, simple, precise and rapid high performance liquid chromatographic method was developed for the determination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC(18) column using a mixture of 15 mM KH(2)PO(4):acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H(3)PO(4). The proposed method was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. The retention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over the concentration range of 50-10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit for meropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference from endogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficiently sensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patients receiving the drug.     

Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection

A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03?M potassium dihydrogen phosphate (pH 6.5)-acetonitrile 65:35 (v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 274 and 370?nm, respectively. Calibration plots were linear over the concentration range 10-1,000?ng?mL(-1) in human plasma, and the lower limit of quantification was 10?ng?mL(-1). Accuracy was between 0.4 and 6.4% and precision was between 3.1 and 7.5%. Amisulpride was sufficiently stable through three freeze-thaw cycles, during storage for 6?h at room temperature, and for 2?months at -22?°C. The method is suitable for the analysis of clinical samples from pharmacokinetic studies.     

Liquid chromatography–electrospray ionization tandem mass spectrometry for the quantification of styraxlignolide A in rat plasma

Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC₁₈ column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r²=0.9978) over the concentration range of 100?10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 μL of plasma sample. The coefficient of variation and relative error for intra‐ and inter‐assays at four QC levels were 1.6–8.3% and from ?12.0 to ?1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague–Dawley rats.     

Bioanalytical technique for determination of tasimelteon in human plasma by LC–MS/MS and its application to pharmacokinetic study in humans

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry technique for the quantification of tasimelteon in human plasma has been developed and validated using tasimelteon‐d₅ as internal standard. Liquid–liquid extraction technique with ethyl acetate was used for extraction of tasimelteon from the plasma. The chromatographic separation was achieved on an Agilent Zorbax, Eclipse, C₁₈ (4.6 × 50 mm, 5 μm) column using a mobile phase of acetonitrile and 0.02% formic acid buffer (85:15, v/v) with a flow rate of 0.5 mL/min. A detailed method validation was performed as per the United States Food and Drug Administration guidelines. The linear calibration curve was obtained over the concentration range 0.30–299 ng/mL. The API‐4000 liquid chromatography–tandem mass spectrometry was operated under multiple reaction monitoring mode during analysis. The validated method was successfully applied to estimate plasma concentration of tasimelteon after oral administration of a single dose of a 20 mg capsule in healthy volunteers under fasting conditions. The maximum concentration of the drug achieved in the plasma was 314 ± 147 ng/mL and the time at which this concentration was attained was 0.54 ± 0.22 h.     

Liquid chromatographic assay for the protease inhibitor atazanavir in plasma

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti-human immunodeficiency virus. A sensitive and selective reversed-phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 microL plasma sample using liquid-liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44-4395 ng/mL atazanavir), intra-day precisions were < or =7% and inter-day precisions were < or =14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng/mL with an intra-day precision of 7%, an inter-day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co-administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV-infected patient treated with atazanavir.     

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry

The interest in therapeutic drug monitoring has increased over the last few years. Inter‐ and intra‐patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield^TM C₁₈ column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease. Copyright © 2015 John Wiley & Sons, Ltd.     

Performance analysis of a reversed-phase liquid chromatographic assay of lamotrigine in plasma using solvent-demixing extraction.

A reversed-phase column liquid chromatographic assay is described and validated for lamotrigine, a new anticonvulsant drug. The drug and its internal standard were extracted from plasma into acetonitrile according to a previously described solvent-demixing procedure, separated on LiChrospher 100CN, and measured by ultraviolet absorption at 280 nm. The assay performance was evaluated through analysis of variance and of regression with our usual validation design. The method detects ca. 2 ng (55 micrograms/l x 30 microliters) and shows a linear response with a constant 5% coefficient of variation from 1 to 10 mg/l. It is easy and robust, and seems well suited to therapeutic drug monitoring.     

Simultaneous quantification of levofloxacin,pefloxacin, ciprofloxacin and moxifloxacin in microvolumes of human plasma using high‐performance liquid chromatography with ultraviolet detection

Levofloxacin, pefloxacin, ciprofloxacin and moxifloxacin are four fluoroquinolones used in the treatment of serious bacterial infections. The antibacterial activity of fluoroquinolones is concentration dependent. Therefore, therapeutic drug monitoring in daily clinical practice is warranted to ensure the therapy's efficacy and prevent bacterial resistance. The purpose of the present study was to develop a method using high‐pressure liquid chromatography with an ultraviolet detector for simultaneous quantification of these four fluoroquinolones in human plasma. A 50 μL aliquot of plasma was precipitated by 200 μL of methanol using gatifloxacin as internal standard. The chromatographic separation was performed on a Kinetex XB‐C₁₈ column using a mobile phase composed of a mixture of orthophosphoric acid 0.4% (v/v), acetonitrile and methanol at a flow rate of 1.2 mL/min. Dual UV wavelength mode was used, with levofloxacin and moxifloxacin monitored at 293 nm, and pefloxacin and ciprofloxacin monitored at 280 nm. The calibration was linear over the ranges of 0.125–25 mg/L for levofloxacin, 0.1–20mg/L for moxifloxacin and 0.05‐10 mg/L for both pefloxacin and ciprofloxacin. Inter‐ and intra‐day trueness and precision were <13% for all the compounds under study. The proposed method was simple, reliable, cost‐effective and suitable for therapeutic drug monitoring or pharmacokinetics studies.     

A novel method for quantification of sulfolane (a metabolite of busulfan) in plasma by gas chromatography–tandem mass spectrometry

The role of busulfan (Bu) metabolites in the adverse events seen during hematopoietic stem cell transplantation and in drug interactions is not explored. Lack of availability of established analytical methods limits our understanding in this area. The present work describes a novel gas chromatography-tandem mass spectrometric assay for the analysis of sulfolane (Su) in plasma of patients receiving high-dose Bu. Su and Bu were extracted from a single 100 μL plasma sample by liquid-liquid extraction. Bu was separately derivatized with 2,3,5,6-tetrafluorothiophenolfluorinated agent. Mass spectrometric detection of the analytes was performed in the selected reaction monitoring mode on a triple quadrupole instrument after electronic impact ionization. Bu and Su were analyzed with separate chromatographic programs, lasting 5?min each. The assay for Su was found to be linear in the concentration range of 20-400?ng/mL. The method has satisfactory sensitivity (lower limit of quantification, 20?ng/mL) and precision (relative standard deviation less than 15?%) for all the concentrations tested with a good trueness (100?±?5?%). This method was applied to measure Su from pediatric patients with samples collected 4?h after dose 1 (n?=?46), before dose 7 (n?=?56), and after dose 9 (n?=?54) infusions of Bu. Su (mean?±?SD) was detectable in plasma of patients 4?h after dose 1, and higher levels were observed after dose 9 (249.9?±?123.4?ng/mL). This method may be used in clinical studies investigating the role of Su on adverse events and drug interactions associated with Bu therapy.     

HPLC with fluorescence detection assay of perampanel,a novel AMPA receptor antagonist,in human plasma for clinical pharmacokinetic studies

Perampanel (Fycompa®), a novel α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial‐onset seizures. To support therapeutic drug monitoring, a simple high‐performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C₁₈ column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile–water–acetic acid–sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0–500 ng/mL) and this could be extended to 25 µg/mL by 50‐fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug‐free blank human plasma from six individuals and no interference was observed with co‐medications tested. Intra‐ and inter‐batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. Copyright © 2015 John Wiley & Sons, Ltd.     

Fast high-performance liquid chromatographic determination of R(+)[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1-H-fluoren-7-yl)oxy]acetic acid in human plasma and urine

A simple and rapid high-performance liquid chromatographic method for the determination of R[(5,6-Dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1-H-fluoren-7-yl)oxy]acetic acid (I) in human plasma and urine is described. The method utilizes Bond-Elut® cartridge facilitate the drug extraction. Analysis is performed on a short reversed-phase column with a mobile phase consisting of acetonitrile and phosphate buffer and quantification is carried out by ultraviolet detection with a wave-length set at 340 nm. The method is linear and reproducible for both plasma and urine analyses (0.25–50) μg/mL) with the detection limit of 125 ng/mL of plasma and urine. Plasma and urine concentrations of / at selected time intervals following I.V. administration of single rising doses are presented.     

Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

A reversed-phase high-performance liquid chromatographic method for the determination of zafirlukast in pharmaceutical formulations and human plasma

Zafirlukast (ZAF) is a leukotriene receptor antagonist used in the treatment of chronic asthma. In this study, a simple and sensitive reversed-phase, high-performance liquid chromatographic method was developed for the determination of ZAF in pharmaceutical formulations and human plasma. Piribedil was used as an internal standard. Analysis was carried out on a Nucleosil C18 100 A (150 mm x 4.6 mm id, 5 Vm) column with acetonitrile-pH 3.0 acetate buffer (70 + 30, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The peak was detected by an ultraviolet detector set at a wavelength of 240 nm. The retention times were about 3.9 min for piribedil and 5.8 min for ZAF. The developed method was applied to the determination of ZAF in its pharmaceutical formulation and spiked human plasma. For quantification of ZAF in spiked plasma, proteins were precipitated with ethanol before chromatographic analysis. The calibration range was linear from 49.69-437.50 ng/mL in spiked plasma. The absolute recovery from spiked plasma was 98.73 +/- 0.42% at a concentration of 254.78 ng/mL of ZAF. No endogenous substances from plasma were found to interfere.     

Development and validation of stability indicating RP-HPLC and HPTLC for determination of Niclosamide in bulk and in synthetic mixture

Two simple, specific, sensitive, accurate and precise stability indicating methods were described for quantitative determination of the anthelmintics drug Niclosamide. The first method was high performance liquid chromatographic with the use of a reversed phase hibar^R C-18 column (250 mm × 4.66 mm, 5 μm) and mobile phase of methanol: 1 mM ammonium phosphate buffer (85:15 v/v) at a flow rate of 1.2 mL/min. The retention time of drug was found to be 6.45 ± 0.02 min. Quantification of drug was achieved with diode array detection (DAD) at 332 nm. Linear calibration curve was obtained in concentration range 0.01–100 μg/mL with r² value of 0.999. The limit of detection and limit of quantification were found to be 0.048 μg/mL and 0.01 μg/ml respectively. The second method involved a high performance thin layer liquid chromatographic. Chromatographic separation was carried out with precoated silica gel G60 F254 aluminum sheets using toluene:ethyl acetate (7:3% v/v) as a mobile phase. Linearity of proposed method was found to be 200–700 ng/band at 332 nm with retention factor of 0.59 and r² value of 0.998. The limit of detection and limit of quantification were found to be 36.21 ng/band and 109.7 ng/band respectively. Both the developed methods were successfully validated as per International Conference on Harmonization guideline (ICH). Niclosamide was subjected to different stress conditions. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention time. Stress samples were successfully assayed by developed high performance liquid chromatographic and high performance thin layer liquid chromatographic method. Statistically analysis proves that there were no statistical significant differences between two developed methods.     

Determination of atazanavir in human plasma by high-performance liquid chromatography with UV detection

A high-performance liquid chromatographic method for the determination of atazanavir (ATV) in human plasma is developed and validated. The method involves a rapid and simple solid-phase extraction (SPE) of ATV using Bond-elut C18 3 mL cartridge. The separation of ATV from internal standard and endogenous components is achieved using an isocratic elution on an octyl column and an UV detector set at 260 nm. The method is linear from 20 to 10,000 ng/mL (mean r2 = 0.9991, n = 10). The observed intra- and inter-day assay precision ranged from 2.2% to 14.7%[at the lower limit of quantitation (LOQ)], whereas accuracy varies between 1.0% and 14% (at LOQ). Mean drug recovery is 80.5% for ATV and 78.4% for IS. The method is found to be precise and accurate, practical enough for therapeutic drug monitoring in routine clinical practice and is applied for the assessment of 24-h ATV plasma concentration-time profiles in HIV-infected pregnant women.