Determination of BAY x 7195, a novel leukotriene D4 antagonist,in human plasma by high-performance liquid chromatography with post-column photo derivatisation and fluorescence detection

Methods to determine plasma concentrations of the leukotriene D₄ antagonist BAY x 7195 by HPLC with post-column photo derivatisation and fluorescence detection are described. Following dilution and centrifugation plasma supernatant is injected onto the HPLC system allowing the selective determination of the drug with a limit of quantitation (LOQ) of 10 μg/l (method A). Sensitivity was further enhanced to a LOQ of 0.6 μg/l by employing solid-phase extraction whereby the analyte concentration in the injection solution was increased (method B). Data on recovery, accuracy and precision of both methods throughout the working range are presented. BAY x 7195 is stable in plasma after repeated freeze-thaw cycles and upon storage at −20°C for at least 13 months. Method A was applied to a clinical study with oral administration of 250 mg BAY x 7195 where ca. 1% of the maximum plasma concentrations still could be accurately and precisely quantified. Method B was employed to determine the drug in plasma after administration of 1 mg as aerosol.     

Analysis of ifosforamide mustard, the active metabolite of ifosfamide, in plasma

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0.45–45 μM and the minimum detectable concentration was 0.45 μmol. The samples were stabilised by addition of semicarbazide and sodium chloride. A patient's plasma sample was analysed by means of the described method. The ifosforamide mustard concentration was 2.3 μM.     

Improved sample preparation for the quantitative analysis of paroxetine in human plasma by stable isotope dilution negative ion chemical ionisation gas chromatography-mass spectrometry

An improved sample work-up and derivatisation procedure for the quantitative determination of paroxetine in human plasma by gas chromatography-negative ion chemical ionisation mass spectrometry is presented. Solvent extraction from plasma samples at alkaline pH was combined with derivatisation to the pentafluorobenzyl carbamate derivative in one step and subsequently analysed without any further purification. Thus, lengthy and time-consuming solvent evaporation steps are avoided to assure high-throughput analysis. Complete validation data are presented. The method is rugged, rapid and robust and has been applied to the batch analysis of paroxetine during pharmacokinetic profiling of the drug.     

Simultaneous measurement of plasma concentrations and 13C-enrichment of short-chain fatty acids,lactic acid and ketone bodies by gas chromatography coupled to mass spectrometry

A new method has been developed for the simultaneous measurement, in a reduced plasma sample, of concentration and 13C-isotopic enrichment of acetic, propionic, butyric, lactic, acetoacetic and beta-hydroxybutyric acids by gas chromatography coupled to mass spectrometry. After plasma deproteinisation, a diethylic extraction and a N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide derivatisation were performed. Both diethyl extraction and derivatisation procedures were optimised using the central composite designs methodology. The optimised method provides good linearity, intra-day and within-day repeatability. Except for beta-hydroxybutyric (49 microM) and acetoacetic acid (5 microM), detection limits were ranging between 0.2 and 0.7 microM allowing uses of this method for colonic metabolism studies.     

Determination of the acylcoenzyme A cholesterol acyltransferase inhibitor 447C88 in plasma using gas chromatography-mass spectrometry and liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry

Two mass spectrometry-based methods are described for the determination of 447C88 (I), a novel inhibitor of acylcoenzyme A cholesterol acyltransferase (ACAT), in rat, dog and human plasma. The first method uses gas chromatography-mass spectrometry (GC-MS) with electron ionisation and selected-ion monitoring. The method employs solid-phase extraction of I from plasma and requires alkylation of I using iodoethane. The second method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical-ionisation and selected-reaction monitoring. The LC-MS-MS method uses a simplified version of the extraction procedure used for GC-MS and does not require derivatisation of I. While both methods provide the necessary limit of quantitation of 0.5 ng/ml in human, dog and rat plasma with the required precision and accuracy, the LC-MS-MS assay offers increased sensitivity, selectivity and speed over the GC-MS assay. This allows a same day turn round of results for in excess of 100 samples, including sample preparation and data acquisition and processing.     

Synthesis of a deuterated analogue and development of antibody/GC/MS for the determination of nocloprost in plasma

An analytical method for the determination of the PGE derivative nocloprost in plasma was developed combining the features of both radioimmunoassay and GC/MS. The antibody usually employed in nocloprost radioimmunoassay was coupled to Sepharose 4B and used as a stationary phase for the extraction of the drug. After appropriate derivatisation, nocloprost was determined by GC/MS in the negative ion-chemical ionisation mode. As an internal standard deuterated nocloprost was synthesized and added to the plasma samples before extraction. The extraction recovery was 94% and the limit of detection was 5 pg/ml. Intra- and interassay precision at 100 pg/ml was calculated as 3.3 and 4.0%, respectively.     

Synthesis of a deuterated analogue and development of antibody/GC/MS for the determination of nocloprost in plasma

An analytical method for the determination of the PGE derivative nocloprost in plasma was developed combining the features of both radioimmunoassay and GC/MS. The antibody usually employed in nocloprost radioimmunoassay was coupled to Sepharose 4B and used as a stationary phase for the extraction of the drug. After appropriate derivatisation, nocloprost was determined by GC/MS in the negative ion-chemical ionisation mode. As an internal standard deuterated nocloprost was synthesized and added to the plasma samples before extraction. The extraction recovery was 94% and the limiy of detection was 5 pg/ml. Intra- and interassay precision at 100 pg/ml was calculated as 3.3 and 4.0%, respectively.     

Performance evaluation of a reversed-phase,high-performance liquid chromatographic assay of valproic acid involving a “solvent demixing” extraction procedure and precolumn derivatisation

As previously shown by others, the antiepileptic drug valproic acid can be assayed in biological fluids by reversed-phase, high-performance liquid chromatography after derivatisation with a bromomethyl aryl ketone through crown-ether catalysis. It is possible to extract the drug directly into acetonitrile, the solvent used for its derivatisation: when an excess of some salt such as NaCl is added to a mixture of plasma and acetonitrile, the organic solvent separates and valproic acid is extracted into it with a high recovery yield. This “solvent demixing” extraction method has shown excellent reproducibility, as well as promising versatility. Derivatisation, still in acetonitrile, using bromomethyl naphthyl ketone and 15-crown-5 allowed us to get rid of the current heating step without markedly increasing the delay of reaction. Chromatography was performed on a C-18 bonded stationary phase with acetonitrile—water as mobile phase, cyclohexanecarboxylic acid as internal standard and ultraviolet spectrophotometric detection. Statistical analysis of results shows 80% recovery of extraction, good linearity and an inter-extract variation coefficient of 4%, the last mainly ascribable to chromatographic measurements. Recovery is readily improved by increasing the amount of acetonitrile, which was equal to that of plasma in our experiments, since the high sensitivity of detection can tolerate the resulting decrease of valproic acid concentration in the extract.     

Automated gas chromatographic assay for amlodipine in plasma and gingival crevicular fluid

This paper describes an automated capillary gas chromatographic method for the determination of amlodipine in plasma, and in sub-microlitre volumes of gingival crevicular fluid (GCF), in order to assess if amlodipine is present in GCF under conditions of gingival overgrowth, as has been shown for nifedipine, another dihydropyridine drug. Liquid-liquid extraction followed by derivatisation was employed to isolate amlodipine and render it suitable for gas chromatography. Amlodipine was analysed in plasma and GCF of four patients undergoing amlodipine therapy for cardiovascular disorders, three of whom had significant gingival overgrowth. Amlodipine was detected in the plasma of all patients and in massive concentrations in the GCG of those patients with overgrowth, 23- to 290-fold greater than in their plasma. Like nifedipine, amlodipine sequestration into GCF appears to be linked with gingival overgrowth.     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

Synthesis of d1-N-ethyltramadol as an internal standard for the quantitative determination of tramadol in human plasma by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) assay for the determination of tramadol in human plasma is presented. The synthesis of an N-ethyl analogue of the drug is described and its use as an internal standard for the quantitative measurement of tramadol in human plasma is described. The method involves extraction at plasma pH and analysis of the underivatized drug by gas chromatography-electron ionization mass spectrometry using m/z 58 and 73 for detection of tramadol and internal standard, respectively. The calibration curve was linear in the range of 5-640 ng/ml plasma (r=0.9999). The method was validated in the abovementioned calibration range. Data on solution stability, long- and short-term stability of tramadol in plasma samples, freeze-thaw-stability, as well as inter- and intra-day precision and accuracy have been evaluated and are presented. The application of the method to the pharmacokinetic profiling of the drug is demonstrated.     

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma

A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at lambda(ex)=267 nm and lambda(em)=443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 microg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.     

Determination of l-arginine and NG, NG - and NG, NG' -dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives

A new HPLC assay for the detection of L-arginine, NG, NG-dimethyl-L-arginine (ADMA) and NG, NG' -dimethyl-L-arginine (SDMA) in plasma using the derivatisation reagent AccQ-Fluor (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) is described. The fluorescent derivatives produced are extremely stable enabling routine processing of large numbers of samples. Arginine and its metabolites are extracted from plasma on strong cation exchange (SCX) cartridges with NG-monomethyl-L-arginine (NMMA) as internal standard, derivatised and separated on a C18 column with acetonitrile in 0.1M sodium acetate buffer pH 6. Separation of the stereoisomers ADMA and SDMA was excellent and improvements to the solid phase extraction (SPE) procedure enabled good recovery (>80%) of arginine, ADMA and SDMA. The utility of the method is exemplified by comparison of plasma concentrations of ADMA, SDMA and arginine in healthy volunteers and diabetic/ischaemic patients.     

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70 degrees C. The method has been applied to 150 patient samples as part of a medication adherence study.     

Evaluation of an on-line solid-phase extraction method for determination of almokalant, an antiarrhythmic drug, by liquid chromatography

A fully automated liquid chromatographic method based on a Prospekt solid-phase extraction unit is described for determination of the antiarrhythmic drug almokalant in plasma. The assay comprises solid-phase extraction on a C₂ phase and separation on a C₁₈ column with fluorometric detection. In the original procedure 40 samples a day could be run unattended but by modifying the sequence in the solid-phase extraction process it was possible to increase this number to 70. The method gives an absolute recovery of 92% and a repeatability (C.V.) of 2.9% at 75 nmol/1 of plasma. The limit of quantitation is 2 nmol/1 of plasma (C.V. < 20%). As regards accuracy and precision the performance of the method is as good as the manual method based on liquid-liquid extraction. The Prospekt method is, above all, faster and requires far less manual effort.     

Liquid chromatographic determination of oxcarbazepine and its metabolites in plasma of epileptic patients after solid-phase extraction

A method based on high-performance liquid chromatography with UV detection in combination with solid-phase extraction for sample pretreatment has been developed for the simultaneous analysis of the antiepileptic drug oxcarbazepine and its main metabolites in human plasma. The extraction of the analytes from plasma samples was carried out by means of a selective solid-phase extraction procedure using hydrophilic-lipophilic balance cartridges. The separation was obtained on a reversed-phase column (C(18), 150x4.6 mm I.D., 5 micrometer) using a phosphate buffer-acetonitrile-methanol-triethylamine mixture (final apparent pH* 3.5) as the mobile phase. Under these chromatographic conditions, oxcarbazepine and its metabolites 10,11-dihydro-10-hydroxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine and the internal standard are baseline separated in less than 9 min. The extraction yield values were >94% for all analytes and the precision, expressed by the RSD%, was in the low percentage range. For the entire method, including sample pre-treatment and HPLC determination, the linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.995; the limit of quantitation was 15 ng ml(-1). The method was applied to plasma samples from patients undergoing chronic treatment with oxcarbazepine, both in monotherapy and in polytherapy. Based on the analytical parameters precision, accuracy, limit of quantitation and analysis time the method is suitable for routine application in therapeutic drug monitoring.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

Capillary electrophoresis with contactless conductivity detection for the determination of carnitine and acylcarnitines in clinical samples

A capillary electrophoresis method with contactless conductivity detection was developed for the quantification of carnitine and six acylcarnitines in plasma and urine samples. The running buffer employed consisted of 500 mmol/L acetic acid, 1.0 mmol/L hydroxypropyl-β-cyclodextrin and 0.05% Tween at a pH of 2.6. Under these conditions, the isomeric valproyl- and octanoyl-carnitines could be distinguished. The linearity was in the range from 5.0 to 200.0 μmol/L with correlation coefficients between 0.9992 and 0.9997. The limits of detection were between 1.0 and 3.2 μmol/L. Intra- and inter-day precisions as %RSD were better than 10%. The method allows for direct determination without derivatisation or extraction processes. The method was applied for the quantification of carnitine and acetylcarnitine in plasma pre- and post-exercise, and to measure valproylcarnitine in plasma and urine of patients undergoing valproate therapy.     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.