Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.     

Determination of the anticancer agent CI-980 in plasma by achiral liquid chromatography on a Pirkle-type stationary phase

A high-performance liquid chromatographic assay has been developed and validated for the determination of the anticancer agent CI-980 in plasma samples from pediatric patients. After ether extraction from alkaline plasma, the CI-980 was chromatographed on a (R)-N-(3,5-dinitrobenzoyl)phenylglycine stationary phase using a mobile phase composed of hexane–isopropanol (70:30, v/v) modified with 1% acetonitrile. The method was linear over a 0.25 to 25.00 ng/ml range and the intra- and inter-day coefficients of variation (C.V.s) were less than 15%. The method was applied to the determination of the plasma concentration–time profile for a pediatric patient receiving 3.5 mg/m² of CI-980 per day as a continuous 72-h infusion.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic—mass spectrometric assay

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.     

Validation of a sensitive assay for thiocoraline in mouse plasma using liquid chromatography-tandem mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.     

High-performance liquid chromatographic determination of a potent AII receptor antagonist (DMP 811) in plasma

An HPLC assay for DMP 811, 4-ethyl-2-propyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylic acid (I) in rat and dog plasma has been developed. Compound I was isolated from plasma using a liquid—liquid back extraction procedure. The extraction recovery was greater than 81%. Separation of I from endogenous components in plasma was achieved on an E. Merck C₈ column using a mobile phase of 0.05 M ammonium acetate, brought to pH 3.75 with acetic acid, and acetonitrile (78:22, v/v). The eluent was monitored by fluorescence with excitation and emission set at 235 and 370 nm, respectively. The assay was linear from 2 to 2000 ng/ml. Inter- and intra-day coefficients of variation for the rat-plasma assay ranged from 0.9 to 5.2% (5–2000 ng/ml) and 2.7 to 16.5% (2–2000 ng/ml), respectively. The respective coefficients of variation for the dog-plasma assay were 1.9 to 5.6% and 1.2 to 14.0%. The percent differences from the accuracy results were 12% or less. Using 0.5 ml of plasma for extraction, the minimum quantifiable limit was 2 ng/ml. This method has been used to quantify plasma levels of I in rats or dogs following 3–10 mg/kg i.v. or p.o. doses.     

Liquid chromatographic assay for dicloxacillin in plasma

A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.     

Determination of metformin in human plasma by high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of antihyperglycemic agent metformin in human plasma using a novel sample extraction procedure. Liquid-liquid extraction of metformin and ranitidine (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (50:50, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a silica column (250 mmx4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (25:75, v/v), pH 6. The limit of quantification (LOQ) was 15.6 ng/ml and the calibration curves were linear up to 2000 ng/ml. The mean absolute recoveries for metformin and internal standard using the present extraction procedure were 98 and 95%, respectively. The intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 8.3%.     

Determination of trimethoprim in biological fluids by high-performance liquid chromatography

A rapid, sensitive, and specific high-performance liquid chromatographic assay was developed for the determination of trimethoprim in blood, plasma, and urine using normal-phase (adsorption) chromatography on a microparticulate silica column and UV monitoring at 280 nm. Trimethoprim is selectively extracted from the biological sample matrix at alkaline pH with chloroform, providing nearly quantitative extraction (>95%) and a sensitivity limit of 0.01 to 0.02 μg/ml blood or plasma, without interference from sulfonamides.     

Enzyme-linked immunosorbent assay for human plasma apolipoprotein B

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (+/- SD) plasma concentrations were 89 +/- 28 mg/dl. Significant age and sex related differences in apoB levels were noted.     

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma

A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at lambda(ex)=267 nm and lambda(em)=443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 microg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.     

Determination of polymyxin E1 in rat plasma by high-performance liquid chromatography

A precise and accurate HPLC assay for polymyxin E(1) in rat and dog plasma has been validated. Samples and standards are extracted from plasma with a 96-well C(8) extraction disk plate. Sample extracts are derivatized with dansyl chloride, and polymyxin E(1) derivative is quantitated on a C(8) column by HPLC with fluorescence detection. The assay is linear in the range of 0.050-5.00 micro g/ml for polymyxin E(1). The precision and accuracy of polymyxin E(1) plasma assay was well within the recommended limits set in the FDA Guidance for Bioanalytical Method Validation. Polymyxin E(1) stability in rat and dog plasma for 24 h at room temperature and through three freeze-thaw cycles was demonstrated.     

Determination of cediranib in mouse plasma and brain tissue using high-performance liquid chromatography-mass spectrometry

A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid-liquid extraction. The assay was validated for a 2.5-2500 ng/mL concentration range for plasma, and for 1-2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1-14.3% for plasma and 1.5-9.4% for brain homogenate; between-assay variabilities were 2.4-9.2% for plasma, and 4.9-10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a brain distribution study in mice at an oral dose of 5 mg/kg.     

Effect of ATP sensitive potassium channel modifiers on antinociceptive effect of metoclopramide

Metoclopramide, a prokinetic drug, has been documented to produce antinociceptive response in animal models through opioid pathways. Morphine has been shown to act through ATP sensitive potassium channels (KATP) to produce antinociceptive response. However, such a possibility has not been examined for metoclopramide. The present study investigated this using pharmacological tools. Acetic acid induced abdominal constriction assay procedure was utilized to assess antinociception. The results confirmed that metoclopramide has antinociceptive response. Glibenclamide, a KATP channel blocker, pretreatment antagonized this response. Where as, in minoxidil pretreated animals, metoclopramide elicited an enhanced antinociceptive response. Glibenclamide and minoxidil, which are known KATP channel blocker and opener respectively, interfered with metoclopramide antinociception. These finding are suggestive of a role for KATP channels in metoclopramide antinociception in mice.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Gas chromatography-negative ion chemical ionisation mass spectrometry using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl derivatives for the quantitative determination of methylphenidate in human plasma

A novel electrophoric derivatisation procedure using o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride for the quantitative determination of methylphenidate in human plasma is described. The drug can be quantitatively measured down to 0.006 pg/mL plasma due to the extraordinary sensitivity of the derivatives under negative ion chemical ionisation mass spectrometry. Plasma samples were made alkaline with carbonate buffer and treated with extraction solvent (n-hexane) and reagent solution for 15 min, which, after concentration was measured by GC-NICI-MS. The method is rapid as extraction and derivatisation occur in one single step. A stable isotope labelled internal standard was used. Validation data are given to demonstrate the usefulness of the assay, including selectivity, linearity, accuracy and precision, autosampler stability, aliquot analysis, robustness, and prospective analytical batch size accuracy. The method has been successfully applied to pharmacokinetic profiling of the drug after oral administration.     

Assay of timolol in human plasma using gas chromatography with electron-capture detection

A simple and sensitive gas chromatographic method has been developed for the determination of timolol in plasma using electron-capture detection and propranolol as internal standard. Timolol was extracted using butyl chloride and derivatized using trifluoroacetic anhydride in butyl acetate. The lower detection limit for the assay was found to be 1 ng/ml from 1 ml of plasma. Extracted standards gave within-day precision of 12.55, 9.68 and 3.78% for 1, 20 and 100 ng/ml plasma samples, respectively. A recovery of at least 80% of timolol was found using the extraction method described. The assay was used in a randomized cross-over bioequivalence trial using an oral administration of 20 mg of timolol. Pharmacokinetic parameters compare favourably with other literature values.     

Sensitive gas chromatographic method for the determination of cinnarizine and flunarizine in biological samples

A sensitive method has been developed for the determination of the vasoactive compounds cinnarizine and flunarizine in plasma, urine and milk samples from man and animals. The procedure involves the extraction of the drugs and their internal standard from the biological samples at alkaline pH, back-extraction into sulphuric acid and re-extraction into the organic phase (heptane—isoamyl alcohol).The analyses were carried out by gas chromatography using a nitrogen-selective thermionic specific detector. The detection limit was 0.5 ng/ml of biological fluid and extraction recoveries were sufficiently high (87–94%).The method was applied to plasma samples from bioavailability studies of both cinnarizine and flunarizine in healthy volunteers, and to plasma, urine and milk samples from flunarizine-treated dogs.