Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithio-carbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 μl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150×3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 μg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 μg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 μg/ml in plasma (500 μl) using a weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ration values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 μg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Capillary electrophoresis with laser-induced fluorescence detection, an adequate alternative to high-performance liquid chromatography, for the determination of ciprofloxacin and its metabolite desethyleneciprofloxacin in human plasma

A method to determine plasma concentrations of ciprofloxacin and its metabolite desethyleneciprofloxacin (M1) by CE with HeCd laser-induced fluorescence detection is described. Following precipitation of proteins and centrifugation supernatant is injected hydrodynamically (10 s, 0.5 p.s.i.) into the capillary. Overall analysis time for the quantification of both analytes was 7 min. The total amount of plasma needed for multiple injections (n>5) was 10–20 μl. Data on accuracy and precision are presented. The assay performance is compared to the specifications of a validated HPLC method, which is routinely used for the quantification of ciprofloxacin and M1 in body fluids. Both methods showed comparable accuracy and precision for both analytes throughout the whole working range (inter-day precision <9%; inter-day accuracy 96–110%). The limit of quantification (LOQ) of 20 μg/l (M1 10 μg/l) for the CE procedure was slightly higher than for the HPLC method, where 10 μg/l (M1 2.5 μg/l) was determined. However, application of the methods to human plasma samples derived from a clinical study proved that comparable results are obtained and that the sensivity of the HPCE method was sufficient to fully describe typical plasma concentration timie profiles of ciprofloxacin and its metabolite M1. Both the adequate sensitivity and the required smaller sample volume compared to HPLC indicate that the method is feasible for clinical studies where sample amounts are limited, e.g., studies to investigate pharmacokinetics in pediatric patients. Preclinical studies form another possible application of this technique.     

Pharmacokinetics of triclabendazole in rabbits

1.Pharmacokinetic profiles of triclabendazole (TCBZ) following intravenous (i.v.) and oral administration of the drug in rabbits were carried out.2. In normal rabbits, TCBZ was metabolized rapidly to its sulphoxide (TCBZ-SO) and sulphone (TCBZ-SO₂) derivatives following administration, with undetectable concentrations of unchanged TCBZ in the plasma of the treated animals at any time (detection limit, 10 ng/ml).3. The disposition kinetics of this drug in rabbits can be described by a two-compartment open model.4. Mean peak concentrations in plasma of TCBZ-SO and TCBZ-SO₂ of 12.41 μg/ml and 9.5 μg/ml occurred 7.5 and 9.5 hr after oral administration, respectively.5. Both metabolites were eliminated slowly from plasma with elimination half-lives of 16.86 hr for the sulphoxide and 13 hr for the sulphone.6. The area under the plasma concentration versus time curve (AUC) was 240 mg hr/l for the sulphoxide, higher than that found for the sulphone, 185 g hr/l.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

Determination of BAY 12-8039, a new 8-methoxyquinolone, in human body fluids by high-performance liquid chromatography with fluorescence detection using on-column focusing

A reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantification of BAY 12-8039, a new antimicrobially active 8-methoxyquinolone, in biological fluids is described. The method is compared to a microbiological assay (bioassay) based on B. subtilis test strain with a limit of quantification of approximately 60 μg/l. Following dilution and centrifugation, plasma, saliva or urine supernatant is directly injected onto the HPLC system. Concentrations down to a limit of quantification of 2.5 μg/l can be quantified in plasma, saliva and urine. Data on recovery, accuracy and precision of the method throughout the whole working range as well as results on stability of the analyte are presented. The concentration data are correlated with results from the bioassay. BAY 12-8039 is stable in plasma after repeated freeze-thaw cycles and following storage at −20°C for at least 12 months. The results of HPLC measurements excellently agree with bioassay data indicating the relevance of the method as a tool in clinical development to answer pharmacokinetic questions related to antimicrobial activity. The method was applied to human plasma, saliva and urine from subjects after a single oral dose of 400 mg of BAY 12-8039.     

Simple and sensitive high-performance liquid chromatographic method for the determination of an everninomycin,SCH 27899, in rat plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of SCH 27899, an everninomycin antibiotic, in rat plasma. The method involved plasma protein precipation with acetonitrile, followed by reversed-phase HPLC analysis using a polymeric column and a mobile phase containing acetonitrile and ammonium phosphate, pH 7.8. The linear relationship between detector response and concentration was demonstrated with a correlation coefficient of larger than 0.996 at concentrations ranging from 0.2 to 100 μg/ml. The results showed that the HPLC method was accurate (bias ≤6%) and precise (coefficient of variation, C.V.≤6%). The limit of quantitation was 0.2 μg/ml with a C.V. of 2.6% and bias of 5%. SCH 27899 was stable in rat plasma at −20°C for at least 40 days. The HPLC method has been utilized for the determination of SCH 27899 in plasma samples from rats following single intravenous administration (3 mg/kg).     

Leishmania donovani: Oral efficacy and toxicity of formycin B in the infected hamster

Formycin B, a structural analog of inosine, was evaluated as an orally administrable antileishmanial agent. Against Leishmania donovani in hamsters, it achieved an 85–92% reduction in numbers of parasites in livers of infected animals after oral administration at 13 mg/kg/day for 4 days. Its efficacy by oral administration was approximately four to eight times that by intramuscular administration and four times that of the positive control drug Glucantime by intramuscular administration. The levels of formycin B in serum after the final oral administration of 26 mg/kg/day were 1.4 μg/ml at 1 hr and 0.3 μg/ml at 2 hr. The concentration in liver was greater (9.0 μg/ml at 1 hr) and declined more slowly. With this latter dosage or with 104 mg/kg/day there was no acute toxicity of formycin B to bone marrow or formed elements of the blood. The only statistically significant toxicity to the liver was a doubling of serum total bilirubin levels. Comparison of the in vivo efficacy of formycin B against L. donovani to the mild acute toxicity of the drug suggests that formycin B has potential as an oral agent against visceral leishmaniasis.     

Sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate (U-63557A), a new,selective thromboxane synthase inhibitor: Intravenous and oral pharmacokintics in dogs and correlations with exsitu thromboxane B2 production

The pharmacokinetics of a new, selective thromboxane synthase inhibitor, sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate were determined for single dose, bolus intravenous injections (1, 3, and 10 mg/kg); for continuous 24 hr infusions (10 and 30 μg/kg/min); and for oral doses of gelatin encapsulated powdered drug (3, 10, and 30 mg/kg). Drug disappeared biexponentially after intravenous administration, and plasma concentrations were proportional to the dose. Absorption of drug occurred rapidly after its oral administration; peak plasma levels occurred 1–2 hours after ingestion, and circulating drug was detectable within 30 minutes. For all experiments, inhibition of cellular thromboxane B₂ production, $\text{ex situ}$, corresponded with plasma drug levels and its reactivation corresponded with disappearance of the drug indicating that it was not accumulated by platelets.     

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of acetylsalicylic acid (ASA) and its main metabolite salicylic acid (SA) in human plasma. Acidified plasma is deproteinized with acetonitrile which is separated from the aqueous layer by adding sodium chloride. ASA and SA are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: Novapak C₁₈ 4 μm silica,150×4mm I.D.; eluent: 740 ml water, 900 μl 85% orthophosphoric acid, 180 ml acetonitrile) and photometric detection (237 nm). 2-Methylbenzoic acid is used as internal standard. The method allows the determination of ASA and SA in human plasma as low as 100 ng/ml with good precision (better than 10%). The assay was used to determine the pharmacokinetic parameters of ASA and SA following oral administration of 100–500 mg ASA in healthy volunteers.     

Comparison of Active Drug Concentrations in the Pulmonary Epithelial Lining Fluid and Interstitial Fluid of Calves Injected with Enrofloxacin,Florfenicol, Ceftiofur,or Tulathromycin

Bacterial pneumonia is the most common reason for parenteral antimicrobial administration to beef cattle in the United States. Yet there is little information describing the antimicrobial concentrations at the site of action. The objective of this study was to compare the active drug concentrations in the pulmonary epithelial lining fluid and interstitial fluid of four antimicrobials commonly used in cattle. After injection, plasma, interstitial fluid, and pulmonary epithelial lining fluid concentrations and protein binding were measured to determine the plasma pharmacokinetics of each drug. A cross-over design with six calves per drug was used. Following sample collection and drug analysis, pharmacokinetic calculations were performed. For enrofloxacin and metabolite ciprofloxacin, the interstitial fluid concentration was 52% and 78% of the plasma concentration, while pulmonary fluid concentrations was 24% and 40% of the plasma concentration, respectively. The pulmonary concentrations (enrofloxacin + ciprofloxacin combined) exceeded the MIC₉₀ of 0.06 μg/mL at 48 hours after administration. For florfenicol, the interstitial fluid concentration was almost 98% of the plasma concentration, and the pulmonary concentrations were over 200% of the plasma concentrations, exceeding the breakpoint (≤ 2 μg/mL), and the MIC₉₀ for Mannheimia haemolytica (1.0 μg/mL) for the duration of the study. For ceftiofur, penetration to the interstitial fluid was only 5% of the plasma concentration. Pulmonary epithelial lining fluid concentration represented 40% of the plasma concentration. Airway concentrations exceeded the MIC breakpoint for susceptible respiratory pathogens (≤ 2 μg/mL) for a short time at 48 hours after administration. The plasma and interstitial fluid concentrations of tulathromcyin were lower than the concentrations in pulmonary fluid throughout the study. The bronchial concentrations were higher than the plasma or interstitial concentrations, with over 900% penetration to the airways. Despite high diffusion into the bronchi, the tulathromycin concentrations achieved were lower than the MIC of susceptible bacteria at most time points.     

Simultaneous determination of the new anticancer agent amidox and its metabolites in rat bile and plasma by high-performance liquid chromatography

A new reversed-phase ion-pair high-performance liquid chromatography method was developed to study the first-pass hepatic metabolism of the anti cancer drug amidox in bile. Separation of the metabolites was achieved on a Spherisorb C₁₈ column after liquid-liquid extraction using a linear gradient system of heptanesulfonic acid in potassium phosphate monobasic (pH 4.0) with increasing amounts of methanol (0–40%). The method was further applied to a pharmacokinetic study of amidox in rats after 200 mg kg⁻¹ intraperitoneal administration. Using 50 μl of rat bile and 300 μl of rat plasma the limit of detection for amidox was 60 ng and 85 ng, respectively, and the assay was linear from 0.1 to 150 μg ml⁻¹. This method appears to be sensitive enough to be used in further pharmacokinetic studies of amidox in human volunteers.     

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE₄ release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F_1α (6-keto PGF_1α) release and enhanced (36%) the quantities of LTE₄ detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC₅₀ values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE₄ release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF_1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.     

Determination of a new oral cephalosporin,S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C₁₈ analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C₁₈ analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09–9 μg/ml and 0.5–100 μg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, d-Trp7,9, mePhe8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, d-Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

A simple method for the assay of colistin in human plasma, using pre-column derivatization with 9-fluorenylmethyl chloroformate in solid-phase extraction cartridges and reversed-phase high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of colistin in human plasma. Derivatization with 9-fluorenylmethyl chloroformate was performed in the same solid-phase extraction C₁₈ cartridge used for sample pre-treatment, followed by reversed-phase HPLC with fluorimetric detection. Quantification was achieved using the ratio of the summed peak areas of colistin A and B derivatives to that of the derivative of netilmicin (internal standard). Linear calibration curves were obtained within the concentrations of colistin sulfate from 0.10 to 4.0 mg/l in plasma. Accuracy was within 10% and reproducibility (RSD) was less than 10%.     

Simple and rapid quantification of gadolinium in urine and blood plasma samples by means of total reflection X-ray fluorescence (TXRF)

A simple and rapid method to determine gadolinium (Gd) concentrations in urine and blood plasma samples by means of total reflection X-ray fluorescence (TXRF) was developed. With a limit of detection (LOD) of 100 μg L(-1) in urine and 80 μg L(-1) in blood plasma and a limit of quantification (LOQ) of 330 μg L(-1) in urine and 270 μg L(-1) in blood plasma, it allows analyzing urine samples taken from magnetic resonance imaging (MRI) patients during a period of up to 20 hours after the administration of Gd-based MRI contrast agents by means of TXRF. By parallel determination of the urinary creatinine concentration, it was possible to monitor the excretion kinetics of Gd from the patient's body. The Gd concentration in blood plasma samples, taken immediately after an MRI examination, could be determined after rapid and easy sample preparation by centrifugation. All measurements were validated with inductively coupled plasma mass spectrometry (ICP-MS). TXRF is considered to be an attractive alternative for fast and simple Gd analysis in human body fluids during daily routine in clinical laboratories.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of sinefungin, a new antiprotozoal drug, in rat plasma has been developed and validated. Sample preparation was performed at 4°C by deproteinization with acetonitrile. Vidarabine was used as an internal standard. Both sinefungin and vidarabine were separated on a C₁₈ column with a mobile phase of ammmonium dihydrogenphosphate-acetonitrile (95:5, v/v) and detected by ultraviolet absorbance at 260 nm. Recoveries of sinefungin from plasma were 75 ± 3.2% and 81 ± 4.8% following dosage at concentrations of 10 μg/ml and 30 μ/ml, respectively. Using 25- μl of rat plasma the limit of quantitation was 1 μg/ml sinefungin, and the assay was linear from 1 to 30 μg/ml. This method appears sensitive enough to be used in further pharmacokinetic studies of sinefungin in animal models.     

Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column. The limit of detection was 50 ng/ml for 7-hydroxycoumarin and 200 ng/ml for coumarin and 7-hydroxycoumarin glucuronide. The linear range was 0.5–100 μg/ml for each of the analytes. The percentage relative standard deviation about the mean measured concentrations were all below 10%. There was no statistical difference between the standard curves prepared in plasma or serum. The method developed was applied to the determination of each of the three compounds in serum, after the administration of 7-hydroxycoumarin, and in plasma after the administration of coumarin. The concentrations of total 7-hydroxycoumarin in the serum samples were also determined by another HPLC method and the results were compared. There was no statistical difference between the results determined.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.