Klaus Richter

Determination of drugs in biological fluids by high-performance liquid chromatography with on-line sample processing

An automated two column HPLC system with the new packing material LiChrospher RP-18 ADS (alkyl-diol-silica) was tested for the determination of several drugs and metabolites (talinolol, celiprolol, metoprolol, oxprenolol, triamterene, trimethoprim, tiracizine, articaine, detajmium, ajmaline, lamotrigine) in various biological fluids (serum, urine, intestinal aspirates, supernatants of cell cultures and supernatants after protein denaturation). The method allows the direct injection of biological fluids into a reversed-phase HPLC system and on-line clean-up and sample enrichment by a column-switching technique. Precision, accuracy and sensitivity were similar to conventional assays as described in the literature. With this new method it was possible to measure drug concentrations in various biological fluids without changing the sample preparation procedure. In some cases an additional sample preparation like protein denaturation or solid-phase extraction was advantageous to enhance the sensitivity of the method and the life-time of the ADS column.

New sensitive method for the determination of hydrochlorothiazide in human serum by high-performance liquid chromatography with electrochemical detection

A HPLC method with a very sensitive electrochemical detection has been developed for determining the diuretic agent hydrochlorothiazide (HCT) in serum of volunteers to whom a single dose of the fixed combination of 12.5 mg HCT and 25 mg triamterene was administered. In the present method samples (0.2 ml serum, pH 7) were purified by extraction of HCT into 5 ml tert.-butyl-methyl ether. The separated organic phase was spiked with p-aminobenzoic acid (PABA) standard. The separation was performed on a reversed-phase C18 column using phosphate buffer-acetonitrile (90:10). A coulometric cell was used to measure HCT and an ultraviolet detector for PABA. The limit of quantitation for serum samples of only 200 microliters was 5 ng/ml (i.e. 60 pg HCT/injection) with a good reproducibility (1-8%). Short retention times were found: 1.2 min for PABA and 5.8 min for HCT.

Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC-MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.

Elucidation of the structure of talinolol metabolites in man determination of talinolol and hydroxylated talinolol metabolites in urine and analysis of talinolol in serum

The objective of this study was to determine the structure of talinolol metabolites formed and the amounts excreted in urine. Talinolol metabolites in urine were identified by comparing their HPLC retention times and their GC-MS profile with those of previously characterized reference compounds. The metabolites were quantified by HPLC with a normal-phase silica column, a single chloroform extraction and UV detection. Less than 1% of an administered dose was found in urine as hydroxylated talinolol. Other metabolites could be excluded. A sensitive method to determine talinolol in serum and a simple method for analysis of talinolol in urine are described. These methods were found to be precise and accurate for the measurement of talinolol in samples obtained from patients during chronic talinolol treatment as well as from healthy volunteers after a single dose of talinolol.

Determination of scopolamine in human serum by gas chromatography-ion trap tandem mass spectrometry

The objective of this study was to develop a very sensitive and selective method for the determination of scopolamine in serum with a rapid and simple sample preparation. A capillary column gas chromatographic-ion trap tandem mass spectrometric technique has been applied. Scopolamine and the internal standard mexiletine were extracted from serum samples and cleaned up by using a single step liquid-liquid extraction. Derivatization was carried out using 2,2,2-trifluoro-N-methyl-N-trimethylsilylacetamide. The mass spectrometer was operated with positive ions in the selected reaction mode with chemical ionisation using methane. The sum of peak height of two daughter ions was used for quantification. The detection limit was 50 pg/ml in serum.

Pharmacokinetics of Articaine Hydrochloride in Tumescent Local Anesthesia for Liposuction

The aim of the investigation was to assess the pharmacokinetic characteristics and safety of articaine HCl used in tumescent local anesthesia for liposuction. Maximum plasma concentrations of articaine HCl were observed from 136 to 264 ng/mL, on average, from 1.2 to 4.3 hours after the start of infiltration, depending on the area of liposuction. The average extent of absorption ranged from 827 to 2203 ng•h/mL. Average maximum plasma concentrations of articainic acid ranged from 1719 to 7292 ng/mL. The high articainic acid concentrations at 1 hour after the start of infiltration indicate that articaine HCl was hydrolyzed rapidly by esterases in tissue and plasma. Although up to 38.2 mg/kg body weight articaine HCl was applied, no cardiac side effects or symptoms of central nervous intoxication occurred. Articaine HCl provided a safe and sufficient analgesia for tumescent liposuction.

Determination of clozapine in human serum by capillary gas chromatography

A gas chromatographic method using a fused-silica wide-bore capillary column and a nitrogen-specific detector for the determination of the antipsychotic agent clozapine in human serum is described. This method was found to be suitable for the determination of serum levels down to 1-2 ng/ml. The sensitivity, precision and accuracy of this method are adequate for studies on pharmacokinetics and bioavailability.

Effect of the Hypoxic Cell Sensitizer Isometronidazole on Local Control of Two Human Squamous Cell Carcinomas after Fractionated Irradiation

Background and Purpose:Hypoxia of clonogenic tumor cells is a major reason for radioresistance and hence local failure in radiotherapy. The objective of the present study was to test the efficacy of the hypoxic cell sensitizer isometronidazole (ISO) during fractionated irradiation in two different human squamous cell carcinomas. Material and Methods:Local control was evaluated for FaDu (radiobiological hypoxic fraction [rHF] 7%) and GL tumors (rHF 0.1%) after single-dose (SD) irradiation under ambient conditions and after 30 fractions within 6 weeks (30 f/6 w). ISO was applied 60 min before SD irradiation at a concentration of 100 mg/kg body weight (b.w.) or 750 mg/kg b.w. in both tumors. During fractionated irradiation, ISO was applied daily 60 min before each fraction (100 mg/kg b.w., in FaDu also 750 mg/kg b.w.).Results:100 mg/kg b. w. ISO did not improve local control after SD irradiation or 30 f/6 w in both tumor models. Application of 750 mg/kg b. w. ISO significantly decreased the SD-TCD50 in FaDu tumors (dose-modifying factor [DMF] = 1.2; p = 0.01) but not in the better oxygenated GL tumor. ISO at 750 mg/kg b.w. also significantly improved local control of FaDu tumors after 30 fractions in 6 weeks (DMF = 1.2; p = 0.01), indicating that hypoxic clonogenic cells in FaDu tumors are not only present before start of irradiation but also limit the efficacy of treatment during a fractionated course of radiotherapy.Conclusion:ISO at a concentration of 750 mg/kg b.w. shows an efficacy as a hypoxic cell sensitizer in severely hypoxic FaDu tumors but not in less hypoxic GL tumors. This supports the principle of hypoxic cell sensitization and improvement of local control of hypoxic tumors by nitroimidazole derivatives. However, doses of 750 mg/kg b. w. before each fraction may be difficult to achieve in the clinical situation. This, in light of the fact that other well-tolerable hypoxic cell sensitizers such as nimorazole with clinically proven efficacy at daily oral doses of < 3 g are available, limits the potential usefulness of ISO for radiation oncology.Hintergrund und Ziel:Hypoxie klonogener Zellen ist eine wichtige Ursache für die Strahlenresistenz von Tumoren und damit für Lokalrezidive nach Strahlentherapie. Das Ziel der vorliegenden Studie war die Überprüfung der Effektivität des „hypoxic cell sensitizer“ Isometronidazol (ISO) während einer fraktionierten Strahlentherapie in zwei unterschiedlichen menschlichen Plattenepithelkarzinomen.Material und Methodik:Die lokale Kontrolle wurde für FaDu- (radiobiologische hypoxische Fraktion [rHF] 7%) und GL-Tumoren (rHF 0,1%) nach Einzeldosis-(SD-)Bestrahlung unter normalen Blutflussbedingungen und nach 30 Fraktionen in 6 Wochen (30 f/ 6 w) bestimmt. ISO wurde bei beiden Tumoren 60 min vor der SD-Bestrahlung in einer Konzentration von 100 mg/kg Körpergewicht (KG) oder 750 mg/kg KG appliziert. Während der fraktionierten Bestrahlung erfolgte die Applikation von ISO täglich 60 min vor jeder Fraktion (100 mg/kg KG, bei FaDu zusätzlich 750 mg/kg KG).Ergebnisse:In beiden Tumormodellen konnte weder nach SD-Bestrahlung noch nach fraktionierter Bestrahlung eine Verbesserung der lokalen Tumorkontrolle durch die Applikation von ISO 100 mg/kg KG gezeigt werden. 750 mg/kg KG ISO führten zu einer signifikanten Reduktion der SD-TCD50 bei FaDu-Tumoren (dosismodifizierender Faktor [DMF] = 1,2; p = 0,01), aber nicht bei den besser oxygenierten GL-Tumoren. ISO in einer Dosis von 750 mg/kg KG führte auch zu einer signifikanten Verbesserung der lokalen Kontrolle von FaDu-Tumoren nach 30 Fraktionen in 6 Wochen (DMF = 1,2; p = 0,01). Dies deutet darauf hin, dass FaDu-Tumoren nicht nur vor Beginn der Behandlung hypoxische klonogene Zellen enthalten, sondern dass diese auch während einer fraktionierten Bestrahlung die Effizienz der Therapie limitieren.Schlussfolgerung:ISO zeigt in einer Konzentration von 750 mg/kg KG eine Effektivität als „hypoxic cell sensitizer“ im ausgeprägt hypoxischen FaDu-Tumor, aber nicht im weniger hypoxischen GL-Tumor. Diese Daten unterstützen das Prinzip der Strahlensensibilisierung und Verbesserung der lokalen Kontrolle hypoxischer Tumoren durch Nitroimidazolderivate. Die zur Radiosensibilisierung notwendigen ISO-Dosen vor jeder Fraktion sind jedoch in der klinischen Situation schwierig zu erreichen. Die Tatsache, dass gut verträgliche „hypoxic cell sensitizer“ wie Nimorazol, deren Wirksamkeit bei täglichen Dosen von < 3 g per os bereits klinisch nachgewiesen wurde, zur Verfügung stehen, limitiert den potentiellen Nutzen von ISO für die Radioonkologie.

Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum

A new method is described using solid-phase extraction (SPE) for preconcentration of articaine and the metabolite articainic acid and high-performance liquid chromatography (HPLC) for the determination of both compounds in human serum. Articaine and articainic acid were extracted in one step with SDB-RPS disk cartridges after precipitation of the serum proteins by perchloric acid. The HPLC separation was then performed on a reversed-phase C8 column using phosphate buffer-acetonitrile (88:12, v/v). UV absorption at 274 nm was used for measuring the analytes with a low limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies of low dose submucosal injections of the local anaesthetic agent articaine hydrochloride in dentistry.

High-performance liquid chromatographic determination of propiverine and its N-oxide in human serum

A high-performance liquid chromatographic method has been developed for the determination of propiverine hydrochloride (P4) and its main metabolite, propiverine N-oxide (P4NO) in human serum. P4 has been shown to be efficacious in those patients who have either idiopathic bladder instability, or neurogenic bladder (detrusor hyperflexia) resulting from spinal cord injuries. In the present method, the analytes were extracted from serum (1 ml, pH 8) into methyl tert.-butyl ether. The separation was performed on a reversed-phase C8 (RP-select B) column using phosphate buffer-acetonitrile (30:70, v/v). UV absorption was used for measuring the analytes, with a limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies.