Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Method for the biological monitoring of hexahydrophthalic anhydride by the determination of hexahydrophthalic acid in urine using gas chromatography and selected-ion monitoring

A method for the determination of hexahydrophthalic acid, a metabolite of hexahydrophthalic anhydride, in human urine has been developed. The urine was worked-up by liquid—solid extraction, esterified with boron trifluoride—methanol, and analysed by capillary gas chromatography and selected-ion monitoring. Hexadeuterium-labelled hexahydrophthalic acid was used as the internal standard. The precision was 4% at 0.7 μg/ml and 5% at 0.07 μg/ml. The recovery of the acid for the overall method was 101% at 0.07 μg/ml of urine (with a coefficient of variation of 4%) and 95% at 0.7 μg/ml (coefficient of variation 2%). The limit of detection was 20 ng/ml urine.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

Simultaneous determination of zidovudine and its monophosphate in mouse plasma and peripheral red blood cells by high-performance liquid chromatography

Novel prodrugs for the intracellular delivery of zidovudine monophosphate (AZTMP) have recently been designed. To investigate the bioconversion and pharmacokinetic profiles of these compounds, an analytical method for the simultaneous determination of zidovudine (AZT) and AZTMP in mouse plasma and peripheral red blood cells was developed. Mouse whole blood samples were treated with TBAHS, EDTA and NaH₂PO₄, and separated into plasma and red blood cell portions. Samples were processed by solid-phase extraction using Bond Elut C₁₈ cartridges. Chromatography was performed using an Hypersil ODS column and a mobile phase of 2.9% (v/v) acetonitrile and 97.1% (v/v) phosphate buffer, pH 7.50, with UV detection at 267 nm. The average extraction recoveries of AZTMP and AZT in plasma were approximately 85% and 97% over their linear ranges of 0.05–5 μg/ml and 0.125–25 μg/ml, respectively. Extraction recoveries of AZTMP and AZT from peripheral red blood cells averaged 56 and 69% over their linear ranges of 0.125–5 μg/ml and 0.125–25 μg/ml, respectively. The accuracy of the assay was 90–100%. The intra- and inter-day variations of the assay were less than 14%. The analytical method was found to be applicable, reliable and suitable for pharmacokinetic studies.     

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

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Column-switching high-performance liquid chromatographic analysis of BO-2727, a new carbapenem antibiotic, in human plasma and urine by direct injection

A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05–50 μg/ml in plasma and 0.5–100 μg/ml in urine. The limits of quantification for plasma and urine were found to be 0.05 μg/ml and 0.5 μg/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.     

Thin-layer chromatographic determination of indolic tryptophan metabolites in human urine using Sep-Pak C18 extraction

Tryptophan and some of its indole metabolites were separated by thin-layer chromatography, stained with the Van Urk—Salkowski reagent, and quantitated by scanning densitometry. The application of this technique for the detection of the indoles in urine samples, employing Sep-Pak C₁₈ cartridges for extraction, was demonstrated. The proposed method is simple and accurate. The detection limits were 2 μg/ml 5-hydroxytryptophan, 1.75 μg/ml 5-hydroxyindolyl-3-acetic acid, 1.5 μg/ml tryptophan, 0.8 μg/ml indolyl-3-acetic acid, 0.9 μg/ml indolyl-3-butyric acid, 1.75 μg/ml serotonin, and 1.25 μg/ml tryptamine.     

High-performance liquid chromatographic method for the determination of mangiferin, likviritin and dihydroquercetin in rat plasma and urine

The use of reversed-phase high-performance liquid chromatography for the determination of the biologically active plant phenolic compounds mangiferin, likviritin and dihydroquercetin is described. Perchloric acid (35%) was used for deproteinization in the case of mangiferin and likviritin, and acidified methanol for dihydroquercetin. Detection was performed at 254, 275 and 290 nm for mangiferin, likviritin and dihydroquercetin in plasma, and 365, 312 and 290 nm in urine, respectively. The limit of detection was 0.2 μg/ml for plasma and 0.5 μg/ml for urine.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

High-performance liquid chromatographic assay for a new fasciolicide agent, αBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (αBIOF10) — a new fasciolicide agent — and its sulphoxide (SOαBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a μBondapak C₁₈ (10 μm) column, using methanol–acetonitrile–water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 12 ng/ml for αBIOF10 and SOαBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 μg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both αBIOF10 and SOαBIOF10. In urine, recovery was 99.6% and 93.1% for αBIOF10 and SOαBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N₄-acetyl-5-methylhydroxysulfamethoxazole (N₄SOH) and sulfamethoxazole-N₁-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by β-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N₄-acetylsulfamethoxazole (N₄), SOH, N₄-hydroxysulfamethoxazole (N₄OH), N₄SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N₄ 0.10 μg/ml; N₄SOH 0.13 μg/ml; N₄OH 0.18 μg/ml; SOH 0.20 μg/ml; and Sgluc 0.39 μg/ml. In urine the quantitation limits are: N₄ and N₄OH 1.4 μg/ml; S 1.5 μg/ml; N₄SOH 1.9 μg/ml; SOH 3.5 μg/ml; and Sgluc 4.1 μg/ml. The method was applied to studies with healthy subjects and HIV positive patients.     

Rapid determination of tetracycline and lumecycline in human plasma and urine using high-performance liquid chromatography

A rapid and accurate method for the determination of tetracycline in human plasma and urine is presented. Determination of tetracycline in plasma is based on precipitation of plasma proteins with trifluoroacetic acid, followed by injection of the centrifuged plasma sample onto a μBondapak C₁₈ column. Acetonitrile in phosphate buffer pH 2.2 is used as mobile phase. Only tetracycline, and no trace of lumecycline can be detected in plasma and urine after administration of lumecycline, indicating that lumecycline is completely degraded to tetracycline, lysine and formaldehyde in the gastrointestinal tract prior to absorption.Determination of tetracycline in urine was performed by injection of urine diluted with phosphoric acid onto a μBondapak Phenyl column. The precision of determination of tetracycline in plasma, expressed as the relative standard deviation, was < 3% at tetracycline concentrations of 0.05 and 3.7 μg/ml. Urine determinations were made with a precision of < 1.5% at tetracycline concentrations of 0.5 and 6.7 μg/ml.     

Determination of the major metabolite of phentolamine in human plasma and urine by high-performance liquid chromatography

A reversed-phase, high-performance liquid chromatographic method using UV detection is described for the assay of the major metabolite of phentolamine in plasma and urine before or after enzymatic hydrolysis. Plasma is deproteinized with methanol. The sensitivity limit is 200 ng/ml using 150-μl samples. Urine is either diluted with water or purified after enzymatic hydrolysis. Concentrations down to 2–3 μg/ml could be quantified with acceptable precision. This method was applied to plasma and urine samples from subjects given phentolamine.     

Measurement of 5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol) in human plasma and urine by high-performance liquid chromatography

Three high-performance liquid chromatographic methods are described for the detection of the novel antifolate anticancer drug (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol): one with fluorometric detection and two with detection by UV absorbance. An assay for plasma lometrexol using UV detection (288 nm) and reversed-phase chromatography was developed, with a quantitation limit of 0.2 μg/ml and linearity up to 10 μg/ml. This assay was modified for measurement of lometrexol in urine, with a quantitation limit of 2 μg/ml and linearity up to 25 μg/ml. An alternative assay for plasma lometrexol using derivatization and fluorescence detection (excitation at 325 nm, emission at 450 nm) was also developed, which proved twenty-fold more sensitive (quantitation limit of 10 ng/ml) than the UV assay, and which was linear up to 250 ng/ml. The fluoremetric method requires sample oxidation with manganese dioxide prior to analysis, and uses ion-pair chromatography with tetramethylammonium hydrogensulphate as an ion-pair reagent. All assays use a similar preliminary solid-phase extraction method (recovery as assessed by UV absorption >73%), with C10-desmethylene lometrexol added for internal standardisation. Each assay is highly reproducible (inter-assay precision in each assay is <10%). Applicability of the fluorescence-based assay to lometrexol in plasma and the UV-based assay lometrexol in urine is demonstrated by pharmacokinetic studies in patients treated as part of a Phase I clinical evaluation of the drug.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.