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%).     

RP‐HPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma

Abstract

A simple reverse‐phase high‐performance liquid chromatographic method for the determination of tenofovir disoproxil fumarate (TDF) in pharmaceutical formulations and human plasma samples has been developed and validated. Piroxicam (PRX) was used as an internal standard. The assay of the drug was performed on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) with UV detection at 259 nm. The mobile phase consisted of acetonitrile–water mixture in the ratio of 75∶25, and a flow rate of 1 ml/min was maintained. The standard curve was linear over the range of 0.2–10 µg/ml (r ²=0.9966). Analytic parameters have been evaluated. Within‐day and between‐day precision as expressed by relative standard deviation was found to be less than 2%. The method has been applied successfully for the determination of TDF in spiked human plasma samples and pharmaceutical formulations.     

A Rapid High Performance Liquid Chromatographic Method for the Simultaneous Measurement of Six Tricyclic Antidepressants

Abstract

A reversed-phase high pressure liquid chromatographic procedure has been developed for the quantitation of the concentration of six different tricyclic antidepressants in the plasma of patients undergoing routine drug therapy. Plasma samples were extracted into a 97:3 hexane:isoamyl alcohol solution and then extracted back into dilute acid. A Supelcosil C-8 column with 5 micron packing was employed in combination with an acetonitrile/ phosphate buffer/diethylamine mobile phase. At an optimized mobile phase pH of 7.22, baseline separation of all six tricyclic antidepressants plus the internal standard was achieved within 8 minutes. UV detection at 254 nm resulted in limits of detection of 2.5 μg/L for each drug. The potential interferences from 13 different benzodiazepines and neuroleptics was investigated. Five of the 13 parent drugs and three metabolites were found to interfere with this tricyclic antidepressant assay.     

Quantitation of Cyproheptadine in Plasma and Urine by HPLC

Abstract

A sensitive, reliable and specific high performance liquid chromatographic procedure has been developed for the quantitation of cyproheptadine in plasma or urine. After extraction of the drug with ethyl acetate from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with acetonitrile and chromatographed using a C₈ reversed-phase analytical column with UV detection at 254 nm. The average recoveries of cyproheptadine from spiked plasma and urine samples in the concentrations ranging from 0.2 – 3 mcg/ml were 95.7 and 100.3%, respectively and their respective CV was 4.1 and 3.9%. Regression analyses for the calibration plots for plasma and urine standards obtained on three different days for the drug concentrations between 0.2 – 3 mcg/ml indicated excellent linearity (r > 0.999) and reproducibility (CV < 2.0%, p > 0.01). The limit of sensitivity was 50 ng/ml for both plasma and urine samples. The method was applied to monitor the plasma concentration versus time profile of cyproheptadine following a single bolus IV dose of 1 mg/kg in a dog.

Urine samples taken from a human subject for the duration of 24 hours following a single oral dose of 8 mg showed that the cumulative amount excreted in urine as cyproheptadine was approximately 1% of the dose.     

Determination of etretinate and its main metabolite in human plasma using normal-phase high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of astemizole and its primary metabolite in plasma and animal tissues. Both compounds and the internal standard were extracted from alkalinized plasma with heptane--isoamyl alcohol and analyzed using a reversed-phase column and UV monitoring at 254 nm. The detection limits for both compounds were 1 ng/ml of plasma and 5 ng/g of tissue and extraction recoveries were sufficiently high (71-84%). The method was applied to plasma and tissue samples from dogs after repeated oral administration, and to plasma samples from a volunteer taking a 300-mg oral dose of the drug. The results were compared with those obtained by a formerly developed radioimmunoassay.     

Determination of zonisamide by a coated monolithic column

In this paper, a monolithic ODS-silica gel column dynamically coated with cetylpyridinium chloride (CPC) was used to demonstrate the high-speed and efficient separation of zonisamide (1,2-benzisoxazole-3-methanesulfonamide, ZNS), its raw material (1,2-benzisoxazole-3-methanecarbonic acid) and intermediate (sodium 1,2-benzisoxazole-3-methanesulfonate) in drugs. Using a 40 mmol/L sodium perchlorate solution (pH 7.0) containing 10% acetonitrile as eluent, the analytes were eluted with a sharp and symmetrical peak within 3.0 min, detected with UV detection at 210 nm. The column demonstrates excellent stability over time, and exhibits unusual selectivity for pharmaceutical analysis. Thus, by this developed method, zonisamide in drug samples can be determined rapidly with high recoveries and good selectivity.     

Spectrofluorometric determination of fluvoxamine in dosage forms, spiked plasma, and real human plasma by derivatization with fluorescamine

A sensitive, simple, and selective spectrofluorometric method was developed for the determination of fluvoxamine (FXM) in pharmaceutical formulations and biological fluids. The method is based upon the reaction between the drug and fluorescamine in borate buffer of pH 8.0 to yield a highly fluorescent derivative that is measured at 481 nm after excitation at 383 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The method was applied for the determination of the drug over the concentration range of 0.1-1.1 microg/mL with a detection limit of 0.01 microg/mL (2 x 10(-8) M). The proposed method was successfully applied to the analysis of commercial tablets. The results obtained were in good agreement with those obtained using a reported spectrophotometric method. The method was applied for the determination of FXM in spiked human plasma with recovery (n=4) of 97.32 +/- 1.23%, while that in real human plasma (n=3) was 90.79 +/- 2.73%. A proposal for the reaction pathway is presented.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

Spectrofluorimetric determination of acetaminophen with N-bromosuccinimide

A simple, sensitive, and selective method for determination of acetaminophen based on its oxidation using N-bromosuccinimide (NBS) to produce a highly fluorescent product. Optimization of reaction variables was carried out concerning NBS concentration, pH, temperature, reaction time, and stability time. Under optimal analytical conditions, the fluorescent intensity was measured at lambda emission. 442 nm (excitation at lambda 330 nm). The linearity range is 120-800 ng/mL with lower detection limit of 33.6 ng/mL acetaminophen. The method was applied successfully to the determination of the compound in pharmaceutical preparations, with average recovery of 100.3 +/- 2%. The method was also applied successfully to the determination of the drug in spiked plasma samples, with an average recovery of 101.2 +/- 1%. Interference effects of some compounds, present in combination with acetaminophen, were studied and the tolerance limits of these compounds were determined.     

A novel validated LC method for quantitation of lopinavir in bulk drug and pharmaceutical formulation in the presence of its potential impurities and degradation products

A simple isocratic liquid chromatographic method was developed for determination of lopinavir from its related impurities and assay for the first time. This method involves the use of a C(8) (Symmetry Shield RP8, 150 x 4.6 mm, 5 microm) column. The method was validated over the range of limit of quantitation (LOQ) to 120% of impurity specification limit and LOQ to 150% of working concentration for assay. The mobile phase consisted of a mixture of 50 mM of potassium phosphate buffer, acetonitrile and methanol in the ratio of 40:50:10. The flow rate was set at 1.0 mL/min with UV detection monitored at 210 nm. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir in pharmaceutical formulations. The method can be conveniently used in a quality control laboratory for routine analysis for assay and related substances as well for the evaluation of stability samples of bulk drugs and pharmaceutical formulations.     

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.     

High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

Determination of Arotinoid Ro 15–0778 and its Metabolite in Plasma by HPLC

Abstract

A method is described for the quantitative analysis in plasma of Ro 15–0778, an arotinoid, (E)-1, 2, 3, 4-tetrahydro-1, 1, 4, 4-tetramethyl-6-(1-methyl-2-phenylethenyl) naphthalene and its metabolite Ro 14–6113, (E)-4- 2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 methylethenyl) phenol. Following a simple extraction, the compounds are determined by reversed-phase high-performance liquid chromatography (HPLC) and detection at 300 nm. The experimental error is below 12% in the concentration range 6–4350 ng/ml. The detection limit is about 2 ng/ml. This method was applied to plasma specimens collected from patients receiving single or multiple dose administration of this compound.     

Determination of Meloxicam in Tablet Formulations by Ultraviolet Spectrophotometry and High-Performance Liquid Chromatography

ABSTRACT

Assay procedures based on UV spectrophotometry and high-performance liquid chromatography (HPLC) have been developed for the determination of meloxicam in tablet formulations. The HPLC method used a reversed-phase C18 column with 0.05M Tris acetic acid buffer - tetrabutylammonium reagent–acetonitrile as eluent, and UV detection at 360nm with isoxicam as the internal standard. The UV method was based on measuring an acidic solution of the drug at 341nm. A comparison was established in terms of linearity, sensitivity, precision, and accuracy. Both methods were simple and rapid. HPLC was more precise and more accurate, the UV technique was slightly faster.     

Automated Solid Phase Extraction and Hplc Analysis of Ibuprofen in Plasma

Abstract

Ibuprofen is a non-steroidal anti-inflammatory drug, widely used in arthritis and other disorders. We describe a high pressure liquid chromatographic (HPLC) method for the analysis of ibuprofen in plasma, using an automated solid phase extraction technique (the Varian AASP^R). In this method ibuprofen was extracted from 0.5 ml of plasma by application to a C₂ extraction cartridge followed by “on line” elution with the HPLC mobile phase (55% acetonitrile / 45% 0.02 H phosphate buffer; pH 3.0), at a flow rate of 1.5 ml/min. The analytical column was a Nucleosil C₁₈ column and the fluorescence detector was set at 253 nm (excitation wavelength) and 300 nm (emission wavelength). Chromatography was complete in less than 10 mins and the limit of detection was 1.3 /μg/ml. The method is linear through the range of 1.0 to 100.0 /μg/ml with a mean correlation coefficient of 0.9964. Absolute recovery of ibuprofen from the spiked plasma samples ranged from 77.8% to 86.5%. The method was shown to be precise within 11% C.V. and accurate to within 8% over the concentration range studied.     

A simple and cost‐effective HPLC‐UV method for the detection of levetiracetam in plasma/serum of patients with epilepsy

A simple, fast and cost‐effective method was developed and validated for the determination of levetiracetam (LEV) in plasma/serum of patients using high performance liquid chromatography (HPLC) with ultraviolet detection. The stability of LEV plasma/serum samples over time and in different blood collection tubes was evaluated. Serum/plasma samples were deproteinized by methanol spiked with the internal standard, gabapentin. HPLC was carried out on a Venusil XBP C₁₈, 250 × 4.6 mm, 5 μm column, at a flow rate of 1.0 mL/min and with mobile phase consisting of 50 mm potassium dihydrogen phosphate–acetonitrile at a pH of 5.5. The UV detector was set at 205 nm and 10 μL was injected. Total runtime was 15 min. Calibration curves were linear (correlation coefficient = 0.999) over a concentration range of 1–60 μg/mL. Relative standard deviation values for both the inter‐day and intra‐day precision and accuracy were <5% for the concentration range. The influence of different collection tubes and the effect of time on the stability of LEV was investigated. These factors may cause inaccuracies owing to drug–protein binding and interference in the matrix. This method is simple, fast, cost‐effective, reliable and accurate with minimal sample preparation for daily routine use in therapeutic drug monitoring.     

Simultaneous Determination of Inorganic Cations and Anion in Medical Electrolytes by Capillary Electrophoresis

Abstract

Capillary electrophoresis (CE) was developed in order to effect rapid, specific determination of potassium, calcium, sodium, and magnesium. Ions (4 cations, 1 anion) were measured continuously via shifting of samples from the inlet electrode to the outlet electrode; moreover, samples were subjected to indirect and direct ultraviolet (UV) techniques, respectively. For the measurement of cations, imidazole (5 mM) functioned as a UV absorber of indirect detection at 214 nm. Consequently, anions can be measured directly by UV at 214 nm. Separation times of cations and Cl^? anions were 10 min and 2 min, respectively; the detection limit of each ion (chloride, magnesium, sodium, calcium, and potassium) was 0.1 µmol/L. This approach is very simple; additionally, measurement time is extremely short. As a result, the proposed method is suitable for routine assay of electrolytes in pharmaceutical preparations.     

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application

Simple, selective and sensitive high‐performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti‐Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C₁₈ end‐capped column (250 × 4.6 mm, 5 μm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5–300 and 150–3000 ng/mL in plasma, and 15–900 and 450–9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.     

High Performance Liquid Chromatographic Determination of Phenacemide in Biological Fluids

Abstract

A sensitive and reliable high performance liquid chromatographic procedure has been developed for the quantitation of phenacemide in plasma or urine. After simple extraction of the drug with ethylacetate from alkalinized samples and evaporation to dryness, the reconstituted extract was chromatographed using a C₈ reversed phase analytical column with UV detection at 254 nm. Regression analyses for the calibration plots obtained on 3 different days for the drug concentrations ranging 1–15 mcg/ml indicated excellent linearity (r >0.999) and reproducibility (CV< 4%, p >0.01). The mean recovery of spiked phenacemide in plasma and urine from the lower limit of quantitation (1 mcg/ml) to 15 mcg/ml was 97.9 and 96.3%, respectively and their respective CV was 3.53 and 2.58%. The method was applied to monitor the plasma vs. time profile of the drug following a single bolus IV dose of 12 mg/kg in a dog.     

High Performance Liquid Chromatographic Determination of Oxamniquine in Plasma Samples

Abstract

A sensitive and reliable liquid chromatographic assay procedure for the quantitation of oxamniquine in plasma or urine was developed. Chromatographic separation was achieved on a reversed-phase phenyl colum using U.V. Detection at 254 nm. The eluting solvent was the mixture of 0.05 M acetate buffer pH 5 and acetonitrile (3:7). With this mobile phase the drug and its external standard were well separated from the interference of the blank samples. The average recovery of oxamniquine from 3 or more replicate dog plasma samples of different concentration (0.125 ? 4.00 μg/ml) was 95.5% and its coefficient of variation was 4.17%. The reproducibility of the assay was confirmed by the analysis of variance test for the slopes of the three standard plots obtained from plasma samples at three different occasions (F=4.2, p > 0.01). The detection limit for plasma samples was approximately 20 ng/ml. The method was applied to measure the plasma level vs, time profile of this drug following a single bolus intravenous dose of 16 mg/kg to a dog.