Quantitative determination of clonazepam in plasma by gas chromatography-negative ion chemical ionization mass spectrometry

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the quantitative analysis of clonazepam in human plasma is described. Clonazepam (M_r = 315) was derivatized by N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. A pre-conditioning procedure involving injection of a silyl-8 and ethyl acetate extraction solution from plasma reduces the interaction between clonazepam-TMS and the analytical system. The routine limit of quantification was set to be 0.25 ng/ml with an injection volume of 2 μl and a sample volume of 1 ml. The signal-to-noise ratio was greater than five. The detection limit for clonazepam can reach 0.1 ng/ml. The isotope clonazepam-d₅ was used as the internal standard.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA_(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA_(R) in certain labs. A fluorescence based receptor binding assay (RBA_(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY^®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA_(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R² = 0.71) with those of the RBA_(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA_(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA_(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA_(F) advantages include the long-term (> 5 years) stability of the BODIPY^®- PbTx-2 and having similar results as the commonly used RBA_(R). The RBA_(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.     

Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure

Recently, it has been reported that a series of prostaglanding F₂-like compounds (F₂-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF_α² is shown to be a potent vasoconstrictor. We describe an improved method for analysing F₂-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C₁₈) and an aminopropyl (NH₂) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F₂-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF_α² as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF_α². However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF_α² when analyzed for the total (sum of free and esterifield)_ F₂-isoprostances. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.     

High-performance liquid chromatographic determination of diazepam in plasma of children with severe malaria

A sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection was developed for the quantification of diazepam in small plasma samples from children with severe malaria. The method involves plasma deproteinization with acetonitrile, followed by liquid–liquid extraction with ethyl acetate–n-hexane. Diazepam was eluted at ambient temperatures from a reversed-phase C₁₈ column with an acidic (pH 3.5) aqueous mobile phase (10 mM KH₂PO₄–acetonitrile, 69:31, v/v). Calibration curves in spiked plasma were linear from 10 to 200 ng (r²≥0.99). The limit of detection was 5.0 ng/ml, and relative recoveries at 25 and 180 ng were >87%. Intra- and inter-assay relative standard deviations were <15%. There was no interference from drugs commonly administered to children with severe malaria (phenobarbitone, phenytoin, chloroquine, quinine, sulfadoxine, pyrimethamine, halofantrine, cycloguanil, chlorcycloguanil, acetaminophen and salicylate). This method has been used for monitoring plasma diazepam concentrations in children with seizures associated with severe malaria.     

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of

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-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate–methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (λ_ex 325 nm, λ_em 435 nm), a limit of detection of 0.1–0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4–0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86–110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p′-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p′-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C₁₈ cartridge. The extraction containing p,p′-DDT and p,p′-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p′-DDT and p,p′-DDE was 0.1 mg kg⁻¹ liver or brain and 0.1 mg l⁻¹ plasma. For six replicate samples at 40, 4 and 0.2 mg kg⁻¹, intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg⁻¹ were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p′-DDT and p,p′-DDE, and linear over the range 0.04–12 mg kg⁻¹ (mg l⁻¹ for plasma). The absolute recoveries of p,p′-DDT and p,p′-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.     

Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO₂ alone, and then the fluoroquinolones were extracted by SFE with supercritical CO₂ containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r ² value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Simultaneous determination of α-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, α-tocopheryl acetate (α-TA) is the main source of vitamin E used to fortify fish feed. α-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of “total α-tocopherol” (α-T) and subtraction of the natively present α-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of α-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of α-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 μg α-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 μg/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 μg/g dry mass. The recovery of α-TA ranged from 97.7 to 100.8% (concentration=2.1 and 20.5 μg/ml, n=6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 μg/ml. This method was routinely applied to determine α-TA and α-, γ- and δ-tocopherol (α-T, γ-T, δ-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.     

High-performance liquid chromatographic assay for the determination of the novel etoposide derivative dimethylaminoetoposide (NK611) and its metabolites in urine of cancer patients

A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C₁₈ column with a mobile phase of acetonitrile-0.05 M KH₂PO₄ (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1–3.4% and 1.9–2.4% for all analytes and the accuracy was 101–107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I–IV in clinical pharmacokinetic investigations.     

High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.     

Liquid chromatographic–mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthiritis. A robust, highly reliable and reproducible liquid chromatographic–mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C₁₈ column and chromatographed with a mobile phase comprised of acetonitrile–1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r²>0.994) over the concentration range 50–1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58–4.0% relative standard deviation and the accuracy is 99.4–107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC–MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.     

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300×3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (λ_ex 230 nm, λ_em 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 μg/ml with a detection limit of 0.2 μg/ml. This assay is suitable for use in clinical studies with etoposide.     

Liquid chromatography–electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 μl) with diethyl ether using 5-pregnen-3β-ol-20-one-16α-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C₈ column (Zorbax-Eclipse, 50×4.6 mm I.D.) using a steep methanol–water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r²=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at −20°C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Simple method for the analysis of tenoxicam in human plasma using high-performance liquid chromatography

A simple, rapid and cost-effective method for the determination of tenoxicam in human plasma is described, using ketorolac as the internal standard. The extraction procedure utilised 5% zinc sulphate and methanol. A nucleosil C₁₈ column and 35:65 acetonitrile-water phosphate buffered mobile phase (pH 2.8) were used, with ultraviolet detection at 355 nm. The assay was linear in the range 40 ng/ml-10 μg/ml, with recovery of extraction ranging from 87 to 102%. The intra- and inter-assay reproducibility had coefficients of variation of 3.9–7.7 and 1.6% respectively. The limit of detection for this method was 40 ng/ml.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.