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 with electrochemical detection for azithromycin in serum and tissues

High-performance liquid chromatographic methods using reversed-phase chromatography and electrochemical detection have been developed for the quantitation of azithromycin in serum and tissues of laboratory animals and humans. Serum sample preparation involved addition of internal standard, alkalinization, and solvent extraction. Tissue sample preparation involved Polytron homogenization in acetonitrile containing internal standard, evaporation of the supernatant, alkalinization of the residue, and solvent extraction. Serum samples were chromatographed on an alkylphenyl-bonded silica column eluted with pH 6.8–7.2 mobile phase with a dual-electrode coulometric detector operated in the oxidative screen mode. Serum and tissue samples were chromatographed on a γRP-1 alumina column with pH 11 mobile phase with a glassy carbon amperometric detector. Recovery of azithromycin was 87% from serum and 85% from tissues. Linear standard curves were prepared in serum over two concentration ranges (0.01–0.20 and 0.20–2.0 μg/ml) and in tissues over several concentration ranges (0.1–2, 1–10, 10–100, and 100–1000 μg/g). In serum and tissues, intra- and inter-assay precision ranged from 1 to 8% and 4 to 11%, respectively. The tissue assay has been applied to liver, kidney, lung, spleen, muscle, fat, brain, tonsil, lymph nodes, eye, prostate and other urological tissues, and gynecological tissues.     

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 analysis of glycoamines in serum

This report describes the development of an HPLC-UV method for studies of glycoamines and glycoamine-like compounds in normal human serum and osteosarcoma patient serum as potential biological markers of cancer. The glycoamines, a newly recognized class of endogenous, low-molecular-mass biopolymers, are conjugates of amino acids and sugar units, containing 5 to 29 amino acid and 1 to 17 sugar units. After ultrafiltration of serum samples, reversed-phase HPLC separation with diode-array detection was used to obtain standard profiles of serum ultrafiltrates below M_r 10 000 in healthy subjects. These highly reproducible profiles utilized two-dimensional peak identification and were used to develop a statistical profile of the major glycoamine peaks in normal serum. This newly developed analytical method was subsequently used to address a key question: whether or not there is a single tumor-specific glycoamine or a family of tumor-specific glycoamines in cancer patient serum. Preliminary results suggest that this method can separate and detect glycoamines and glycoamine-like compounds in various types of cancer patient serum with a high degree of reproducibility on the basis of comparative two-dimensional identification of natural compounds and a panel of synthetic glycoamine analogs. Moreover, the method is useful for following the relative changes in the amount of a given glycoamine over an extended clinical time course. Initial results suggest that a glycoamine or glycoamine-like compound, GA-4.63, may have clinical utility in human osteosarcoma studies.     

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis™ HLB cartridge and were injected into a C₈ Nucleosil column. The HPLC eluent contained acetonitrile–methanol–distilled water–0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1–25 μg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2–17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4°C, rapid degradation of the drug occurred. Frozen at −30°C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The method’s ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.     

High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver

A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection is described for determining the production of the major N-dealkylated metabolite of amiodarone in rat liver microsomes. The principal advantages of this method are its simple sample preparation (protein precipitation by acetonitrile), low detection limit for N-desethylamiodarone (0.05 μmol/l) and relatively short analysis time (16 min). Its analytical applicability is demonstrated by the comparison of the kinetic parameters (maximum velocity and Michaelis—Menten constant) between Sprague-Dawley and Dark-Agouti rats.     

High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes

A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.     

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.     

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE–glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, was essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE–glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.     

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H(3)PO(4) with 0.2% triethylamine mobile phase was employed and separation was achieved with a C(18) analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9+/-0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 microg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 microg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 microg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 microg/ml. Results of the stability study indicated that when frozen at -80 degrees C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.     

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.