Franz Bucheli

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching : I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.

Determination of highly protein bound in plasma using high-performance liquid chromatography and column switching, exemplified by the retinoids

During method development for the determination of either isotretinoin, tretinoin and their 4-oxo-metabolites, or etretinate, acitretin and 13-cis-acitretin in plasma using high-performance liquid chromatography and column switching, recovery problems arose, when undiluted plasma samples were injected directly onto the precolumn. These recovery problems may be due to the strong binding of the retinoids to different plasma proteins. Measures to overcome this strong protein binding, such as variation of the injection solution composition and the purge mobile phase, were systematically investigated. Best recoveries were obtained by diluting of plasma with 9 mM sodium hydroxide-acetonitrile (8:2, v/v) and protein precipitation with ethanol for the isotretinoin and etretinate series, respectively, in combination with the use of a purge mobile phase containing ammonium acetate and 10-20% acetonitrile. Less effective was the use of a longer precolumn or heating of the precolumn.

Sensitive determination of oseltamivir and oseltamivir carboxylate in plasma, urine, cerebrospinal fluid and brain by liquid chromatography–tandem mass spectrometry

This manuscript describes the determination of oseltamivir (OP) and oseltamivir carboxylate (OC) in rat plasma, cerebrospinal fluid (CSF) and brain and in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry. Threefold deuterated OP and OC served as internal standards. Protein precipitation with perchloric acid was followed by on-line solid-phase extraction and gradient separation on a reversed-phase column. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. Run time was 3.6 min. The lower limits of quantification (LLOQ) were 0.1 ng/mL in rat plasma and CSF, 0.5 ng/g in brain and 1 ng/mL in human plasma and urine. Inter-day and intra-day precisions and inaccuracies in rat matrices were below 10.2% and 13.9% (below 19.0% at LLOQ), respectively. Intra-assay precisions and inaccuracies in human matrices were below 11.7% and 8.9%, respectively. The recoveries were close to 100%, and no significant matrix effect was observed. The method was successfully applied to rat study samples.

Determination of oseltamivir (Tamiflu®) and oseltamivir carboxylate in dried blood spots using offline or online extraction

BACKGROUND Using dried blood spots (DBS) for quantitation of the antiviral drug oseltamivir (Tamiflu(®)), an ester prodrug, and its active metabolite oseltamivir carboxylate could provide ethical and logistic benefits. Hence, its feasibility was investigated using a previously developed column-switching LC-MS/MS method. RESULTS Sensitivity, precision and accuracy in DBS were comparable to standard plasma assays. Chemically treated cards provided enhanced ex vivo stability of the ester prodrug in rodent blood. Online extraction was realized using the manual TLC-MS interface or the fully automated Sample Card and Prep system. Rat pharmacokinetic study data showed good correlation between plasma, liquid blood and DBS. CONCLUSION From a bioanalytical perspective, DBS is potentially suited for Tamiflu analysis in animals and humans. Automation of the process by online DBS extraction promises workload reduction and throughput increase.

Determination of mycophenolic acid and its phenyl glucuronide in human plasma, ultrafiltrate, blood, DBS and dried plasma spots

BACKGROUND Analysis of mycophenolic acid (MPA), the active form of the immunosuppressive drug mycophenolate mofetil, and its glucuronide metabolite MPAG is required for therapeutic monitoring and postmarketing clinical studies. Dried blood spots (DBS) and dried plasma spots (DPS) could be alternatives to conventional assays for small-volume sampling and easy shipment. RESULTS A LC-MS/MS method with online SPE was established using stable isotope labeled analytes as internal standards. The quantitation limits were set at 0.1 and 1 µg/ml, for total MPA and MPAG, respectively, in plasma, blood, DBS and DPS, but 100-fold lower for free MPA in ultrafiltrate. Ahlstrom 226 or Whatman FTA(®) DMPK-B cards were well suited for DBS and DPS analyses. CONCLUSION MPA and MPAG were analyzed in human plasma and blood either as liquid or dried on cards with similar assay quality. Care should be taken to avoid back-conversion of an instable acyl glucuronide metabolite to MPA.

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection.

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate-acetic acid-ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate-acetic acid-ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile-methanol-ethanol-2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5-2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4-96.5% for retinol (range 100-1000 ng/ml) and 92.7-96.0% for retinyl palmitate (range 5-1000 ng/ml). Inter-assay precision was < or =5.1% and < or =6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.

Use of direct injection procolumn techniques for the high-performance liquid chromatographic determination of the retinoids acitretin and 13-cis-acitretin in plasma

A previously developed highly sensitive high-performance liquid chromatographic method for the determination of retinoids, using direct injection of large plasma volumes, on-line solid-phase extraction and ultraviolet detection, was improved and fully validated for the determination of acitretin and 13-cis-acitretin in plasma samples. The addition of acetonitrile to improve the recovery was performed on-line by a T-piece, avoiding any cis-trans isomerization which could occur when acetonitrile was added prior to storage in the autosampler. About 30 injections could be made onto one precolumn despite the large injection volume (1 ml of plasma containing the internal standard). Full automation was attained by the use of automated precolumn replacement. In addition, forward- and back-flush purging of the precolumn enhanced the longevity of the analytical column. This consisted of three coupled C18 columns of 125 mm length each. The quantification limit was 0.3 ng/ml, using ultraviolet detection at 360 nm, and the mean inter-assay precision was 3.8% for the two compounds.

Ultra-fast quantitative bioanalysis of a pharmaceutical compound using liquid chromatography-tandem mass spectrometry

This paper describes the ultra-fast determination of a pharmaceutical compound using TurboIonSpray LC-MS-MS on an API 4000 mass spectrometer. Sample preparation consisted of plasma protein precipitation, centrifugation and dilution of the supernatant. The use of small analytical column dimensions (2.1 mm x 10 mm) and high eluent flow rates (up to 2.2 ml/min) in isocratic mode led to a retention time of 9s. A sample-to-sample cycle time of only 10s was achieved by coupling two autosamplers. Partial separation of the drug and its main metabolite could be obtained. The d5-labeled drug used as internal standard compensated for matrix suppression effects. The assay was linear in the concentration range 1-1000 ng/ml, using standards prepared in human plasma. Inter-assay accuracy and precision were 98.5 and 6.2%, respectively. Mean intra-assay accuracy and precision calculated from quality control (QC) samples in human, rat and dog plasma at 3, 30 and 800 ng/ml were 100.8 and 3.8%, respectively. The ultra-fast LC-MS-MS method was successfully cross-validated against a commonly used column-switching LC-MS-MS assay with 2.3 min run time by analyzing real study samples.

Determination of cefetamet and its orally active ester, cefetamet pivoxyl, in biological fluids by high-performance liquid chromatography.

Two different, simple and rapid high-performance liquid chromatographic methods with ultraviolet detection, using a common sample work-up procedure, were developed for the determination of cefetamet, an in vitro active cephalosporin, and its orally absorbed pivaloyloxymethyl ester, cefetamet pivoxyl. After protein precipitation with perchloric acid, plasma samples were analysed on C18 reversed-phase columns with 4 mM perchloric acid-acetonitrile (83:17, v/v) and 0.1 M phosphate buffer (pH 6.5)-acetonitrile (60:40, v/v) as mobile phases for the determination of cefetamet and cefetamet pivoxyl, respectively. Urine samples were diluted with water and analysed in the same manner, using 4 mM perchloric acid-acetonitrile (85:15, v/v). The limits of quantification were 0.2, 0.5 and 20 micrograms/ml for the determination of cefetamet and cefetamet pivoxyl in plasma and cefetamet in urine, respectively. The intra-assay precision was less than or equal to 1.5% for cefetamet and less than or equal to 2.3% for cefetamet pivoxyl. The inter-assay precision for cefetamet was less than or equal to 2.4%. Cefetamet was stable in human plasma when stored at -20 degrees C for three months or at 22 degrees C for 24 h. For the determination of cefetamet pivoxyl, which was extremely unstable in plasma (greater than 70% degradation in 1 h), samples were drawn into vacutainers containing citric acid and immediately added to sodium fluoride. The method for cefetamet was successfully applied to several thousand plasma and urine samples from humans, dogs and rats. No unchanged drug could be detected in human or dog plasma after the administration of cefetamet pivoxyl.