High-performance liquid chromatographic assay for meropenem in serum

High-performance liquid chromatographic procedures have been developed for the measurement of meropenem in serum. The separation was performed on an Ultrasphere XL-ODS analytical column (75×4.6 mm I.D.). The mobile phase consisted of 10.53 mmol/l ammonium acetate-acetonitrile (95:5, v/v) (pH 4). The UV detection was at 298 nm. The quantitation limit both in serum and water was 0.25 μg/ml. The method was validated in serum and aqueous solution over the concentration range 0.25–50 μg/ml. The extraction recovery from serum spiked with meropenem was 99.7±3.4%. The intra- and inter-assay coefficients of variation were below 6%. Stored at −80°C for three months at various concentrations in serum and in aqueous solution, meropenem did not reveal any appreciable degradation. After 24 h, it was also stable at 4°C in serum, aqueous solution and supernatant of extraction but not at room temperature. The stability of the drug was also confirmed in serum after repeated freezing-thawing cycles at −80°C on four consecutive days.     

High-performance liquid chromatographic assay for cefepime in serum

A simple, rapid, specific and sensitive high-performance liquid chromatographic method was developed for the determination of cefepime 1-[[6R, 7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo-[4.2.0] oct-2-en-3-yl]methyl]-1-methylpyrrolidinium hydroxide, inner salt, 7²-(Z)-(O-methyloxime) in human serum. Separation was achieved on a reversed-phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4). Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV absorbance at 254 nm. The lower limit of quantitation of cefepime in plasma was 0.5 μg/ml. The average absolute recovery was 106.2±2.1%. The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient greater than 0.999. The within-day C.V.s for human samples were 4.9 and 2.3% for 1 and 50 μg/ml, respectively. The between-day C.V.s for human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50 μg/ml, respectively. Cefepime was found to be unstable in serum at room temperature. For delayed assay, samples must be stored at −80°C.     

High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissues using electrochemical detection

This paper describes a sensitive HPLC-electrochemical detection analytical method for determining the concentration of the intravenous anesthetic, propofol, in human or rat plasma or serum and a variety of rat tissues. Internal standard and drug are extracted from serum or plasma and other tissues with pentane. 2,6-tert.-Butylmethylphenol is used as internal standard. It includes a novel steam distillation procedure for separating the highly lipophilic propofol from skin and fat. The plasma/serum assay has a precision of 1–4% (C.V.) in the range 10 ng/ml to 1 μg/ml and permits the assay of 5 ng/ml from 0.1 ml of plasma/serum. The tissue procedure allows the estimation of 50 ng/g in 0.1 g of tissue for most of the major organs with less than 2% (C.V.) precision. This assay was used to measure propofol concentrations in plasma/serum and tissue samples in support of a project to develop a physiological pharmacokinetic model for propofol in the rat.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5′-monophosphoric acid (AMP) in a buffer of required pH, 5′-nucleotidase was inhibited with Ni²⁺ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultra-violet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.     

High-performance liquid chromatographic separation and electrochemical detection of cephalosporins

Pulsed amperometric detection (PAD) is useful for detection of cephalosporins following separation on a C₁₈ column using an acetate buffer solvent with a small percentage of organic modifier. Under these conditions, the indirect PAD mode worked better than direct PAD, with IPAD outperforming both. A gradient program was demonstrated that allowed separation and sensitive electrochemical detection of eleven different cephalosporins with widely differing side chain structures. The cephalosporins could be detected to sub-micromolar levels with this separation. Applications of the method for quantitation of pharmaceutical formulations and for monitoring cephalexin in porcine serum were demonstrated. To improve the detectability of cephalexin, an on-column concentration scheme using separate concentration and elution solvents was applied to porcine serum.     

High-performance liquid chromatographic validated assay of doxorubicin in rat plasma and tissues

A specific and selective high-performance liquid chromatography (HPLC) technique that requires few manipulations, and is readily adaptable to analysis for a large series of samples, has been developed for the determination of the concentration of the anticancer drug doxorubicin (DXR) in rat serum and tissues. The biological samples were efficiently deproteinised and resolved from a reversed-phase nucleosil C₁₈ column with fluorescence detection. The validation study of the proposed method was successfully carried out in an assay range of between 5 and 5000 ng/ml and was subsequently implemented in a pharmacokinetic study of DXR in Wistar rats that were treated by intravenous administration of the drug.     

High-performance liquid chromatographic method with electrochemical detection for the analysis of O6-methylguanine

An improved system consisting of a combination of high-performance liquid chromatographic methods with electrochemical detection for the separation and analysis of the DNA adduct O⁶-methylguanine (O6MG) has been developed. This adduct is produced by the interaction of methylating agents with DNA and induces mispairing in the DNA of the target cells. A good separation of modified from unmodified bases is first achieved with an HPLC system using a Partisil 10 SCX column and a salt gradient. A second HPLC step with electrochemical detection and a C18 column is used for farther separation and quantitation of O⁶-methylguanine. This method shows a linear response up to 15 pg of O6MG tested. The lowest amount detected was 0.5 pg of O6MG and is highly reproducible. This method is useful to study DNA damage as a product of cellular metabolism and its effects on the process of carcinogenesis.     

High-performance liquid chromatographic determination of phenylephrine in human serum with coulometric detection

A high-performance liquid chromatographic method for the determination of phenylephrine (PE) in human serum using coulometric detection is described. PE and internal standard, orciprenaline, were extracted from serum by solid-phase extraction and separation achieved on a coupled column system consisting of two C₁₈ cartridge columns (250×4.6 mm I.D. coupled to a shorter 50×4.6 mm I.D. column) using a mobile phase of methanol-50 mM phosphate buffer (pH 3.2; 10:90) at 36°C. Dual electrode coulometric detection was used in the “oxidative screen” mode. Calibration curves were linear over the range 0.3–4 ng/ml with a limit of quantification (LOQ) of 0.35 ng/ml. The method has a greater degree of sensitivity, precision and accuracy compared to previously published methods for PE and is suitable for use in pharmacokinetic and bioequivalence studies in humans.     

High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples

A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocartic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean±S.D.): 3.148±0.259 ng (8.22%), 12.594±0.571 ng (4.53%), and 25.016±0.658 ng (2.63%) with C.V. values in parentheses for n=5. The accuracy of the assay ranged from −7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5±0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.     

High-performance liquid chromatographic analysis with electrochemical detection of biogenic amines using microbore columns

High-performance liquid chromatography with electrochemical detection (HPLC-ED) is a popular method for measuring biogenic amines, owing to its simplicity, versatility, sensitivity, and specificity. Recent developments in microbore column HPLC-ED have been facilitated by miniaturization of solvent delivery, column packing, sample injection and micro-flow cell construction. The aim of this paper is to present an overview of recent developments in microbore column HPLC-ED, in terms of advantages and limitations. This paper covers the recent advancements and important factors of HPLC-ED analysis of biogenic amines using microbore columns. Particular emphasis is placed on applying this technique to microdialysis, for which great sensitive is required. Its potential in future biomedical applications is also discussed.     

High-performance liquid chromatographic assay for pilocarpine in aqueous humor

A high-performance liquid chromatographic assay for pilocarpine has been developed for the determination of pilocarpine in aqueous humor. A structurally similar internal standard is used, and pilocarpine is separated from isopilocarpine under the chromatographic conditions used. A 100μl sample is mixed with an aliquot of internal standard at pH 8.3 and extracted with methylene chloride. The extract is evaporated to dryness and the alkaloids are quaternized with p-nitrobenzyl bromide. Following the quaternization, the sample is evaporated to dryness, washed and diluted with a mobile phase—triethylamine mixture and analyzed by high-performance liquid chromatography using a reversed-phase octadecylsilane column with detection at a wavelength of 254 nm. This is a highly sensitive, reproducible and selective assay for measuring pilocarpine at physiological levels in individual aqueous humor samples.     

High-performance liquid chromatographic assay for amiloride in plasma and urine

A sensitive and simplified high-performance liquid chromatographic procedure has been developed for quantification of amiloride in rabbit plasma, as well as human plasma and urine. Following protein precipitation with perchloric acid, the supernatant was directly injected into a C₁₈ Nucleosil column. The mobile phase consisted of methanol—water (45:55) containing 0.1 M perchloric acid, and the compound was quantitated using a fluorescence detector at excitation and emission wavelengths of 286 and 418 nm, respectively. The average recovery was 97.6%. The calibration curve was linear over the range 2.0–20.0 ng/ml. The limit of detection was 0.5 ng/ml.     

High-performance liquid chromatographic assay with UV detection for measurement of dihydrouracil/uracil ratio in plasma

A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.     

High-performance liquid chromatographic assay using electrochemical detection for the combined measurement of amifostine,WR 1065 and the disulfides in plasma

A high-performance liquid chromatographic (HPLC) method was developed for the combined analysis of the chemoprotective agent, amifostine, its active metabolite, WR 1065, and the (symmetrical and mixed) disulfides of WR 1065 in plasma. These three compounds were quantified by measuring WR 1065 after three different sample pretreatment procedures. During these procedures, amifostine and the disulfides were quantitatively converted into WR 1065, by incubating the sample either at a low pH or in the presence of dithiothreitol, respectively. The resulting amounts of WR 1065 were determined by HPLC with electrochemical detection (Au electrode, +1.00 V). The lower limit of quantitation of WR 1065 was 0.15 μM. The within-day and between-day precision were ≤4.4 and ≤8.2% for WR 1065, ≤4.9 and ≤13.1% for amifostine and ≤8.5 and ≤5.5% for the disulfides, respectively. The within-day and between-day accuracy ranged from 97.2 to 109.8% and from 97.6 to 101.5% for WR 1065, from 88.3 to 110.7% and from 99.4 to 101.5% for amifostine and from 99.2 to 110.2% and from 103.3 to 104.9% for the disulfides, respectively. This method is superior to other described methods due to its simple and relatively rapid analysis of all three compounds in one system. Furthermore, it is at least as sensitive as earlier reported methods for one of the compounds and the application of the gold electrode requires only minor maintenance. Therefore, this method is very suitable for pharmacokinetic studies of amifostine and its metabolites. As an example, the plasma concentrations of amifostine, WR 1065 and the disulfides are shown in a patient after receiving an i.v. dose of 740 mg/m² amifostine.     

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 degrees C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E(1)=200 mV and E(2)=600 mV; guard cell: E(G)=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 microM. The lower limit of quantitation of WR1065 was 0.25 microM. The within- and between-day precisions were < or = 4.3% and < or = 6.0% for amifostine, < or = 4.4% and < or = 3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.     

High-performance liquid chromatographic assay for farnesyl-protein transferase activity with dabsylated peptide

An HPLC assay for farnesyl-protein transferase activity using a dabsylated peptide is described. The substrates used were a synthetic dabsylated nonapeptide, N-dabsyl-l-serinyl-l-methioninyl-l-glycinyl-l-leucinyl-l-prolinyl-l-cysteinyl-l-valinyl-l-valinyl-l-methionine, corresponding to the C-terminal peptide seqeunce of human N-Ras p21 without the N-terminal serine, and farnesyl disphosphate. The product was separated from the substrates on a reversed-phase C₁₈ column, using gradient elution with acetonitrile (0.05% trifluoroacetic acid)-water (0.1% trifluoroacetic acid) and was detected at 436 nm. The addition of the farnesyl group to the peptide was confirmed by MS and NMR. Enzymatic reaction was ascertained from the dependences on time, on the protein of the enzyme source and on the substrates. The reaction was specifically inhibited by l-cysteinyl-l-valinyl-l-valinyl-l-methionine, the tetrapeptide corresponding to the “CAAX” motif. The limit of detection was 2 pmol per 100-μl reaction mixture. The farnesyl-protein transferase activity can quantitatively be measured up to 200 μg cytosolic protein in human liver. This method provides a convenient and quantitative assay for crude materials, such as tissue homogenate from clinical samples, without the use of radioactive probes and large amounts of Ras protein.