Improved gas chromatographic procedure for the determination of clonazepam levels in plasma using a nitrogen-sensitive detector

A gas—liquid chromatographic procedure (GLC) is described for the determination of clonazepam in plasma. The drug is extracted from buffered plasma at pH 9.0 with diethyl ether and then back-extracted into 6 N hydrochloric acid—6 N sulfuric acid (95:5) and hydrolyzed at 100°C to convert the drug into its benzophenone derivative. The benzophenone derivative of flurazepam is added to plasma as an internal reference standard. Drug derivatives are finally extracted from the neutralized aqueous phase and assayed by GLC. The present procedure makes use of a nitrogen-sensitive detector which is more stable and selective than the commonly employed electron-capture procedure. The sensitivity of the detector for clonazepam is 1 ng/ml.     

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5?μmol/l SYTOX Green for 40?min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6?h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617?mg/l 1-pentanol (95?% confidence limits 4,670-9,327) and 12,818?mg/l 1-pentanol (95?% confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.     

Analysis of barbiturates in blood by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for the identification and quantification of barbiturates in blood at therapeutic levels has been developed. An ODS-silica column is used with an eluent of 40% methanol at pH 8.5. The barbiturates are detected at 240 nm. The sample preparation procedure involves extraction of unfractionated blood (100 μl) with hexane—diethyl ether (50:50, v/v) and is very rapid. Talbutal is used as an internal standard.The method has been applied to the determination of five barbiturates (amylobarbitone, butobarbitone, cyclobarbitone, pentobarbitone and quinalbarbitone) in blood after therapeutic doses of the drugs. An application of the HPLC assay to forensic casework is demonstrated.     

A rapid screening procedure for cholesterol and dehydrocholesterol by electrospray ionization tandem mass spectrometry

The mono-(dimethylaminoethyl) succinyl (MDMAES) ester is a new derivative for rapid, mild, and sensitive electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of cholesterol and dehydrocholesterol. It is an order of magnitude more sensitive than the previous most practical alternative, the N-methylpyridyl ether derivative. The MDMAES derivative was used to develop a rapid screening procedure for the biochemical diagnosis of Smith-Lemli-Opitz syndrome (SLOS) by measuring the dehydrocholesterol/cholesterol ratio in plasma (5 microl) and plasma spotted onto filter paper. Details of the synthesis of [25,26,26,26,27,27,27-(2)H7]-7-dehydrocholesterol, used as a standard for quantitation, are included. The measurement of total sterols as MDMAES esters, after base hydrolysis of plasma, afforded a dehydrocholesterol/cholesterol ratio of 0.05-2.95 for SLOS patient samples (n = 5) compared with 0.001-0.003 for normal adult controls (n = 20). Direct hexane extraction of plasma without base hydrolysis enabled the measurement of free sterols with a total sample analysis time of <1 h. The free dehydrocholesterol/cholesterol ratio was 0.10-4.47 for SLOS patient samples (n = 5) and 0.003-0.011 for normal adult controls (n = 20).     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

High-performance liquid chromatographic determination of pipotiazine in human plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of pipotiazine in human plasma and urine. After selective extraction, pipotiazine and the internal standard (7-methoxypipotiazine) are chromatographed on a column packed with Spherosil XOA 600 (5 μm) using a 7:3 (v/v) mixture of diisopropyl ether—isooctane (1:1, v/v) + 0.2% triethylamine and diisopropyl ether—methanol (1:1, v/v) + 0.2% triethylamine + 2.6% water. The eluted compounds are measured by fluorescence detection. The sensitivity of the method was established at 0.25 ng/ml pipotiazine in plasma and 2 ng/ml pipotiazine in urine (C.V. < 5%). The method has been successfully applied to a pharmacokinetic study following a single oral administration of 10 mg of pipotiazine.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection

A rapid method for the determination of olanzapine in plasma using high-performance liquid chromatography with ultra violet detection is described. Olanzapine was extracted from plasma with a mixture of hexane/dichloromethane (85:15), and then back extracted into phosphate buffer pH 2.8. Separation was achieved on a RP Select B C(18) column and commonly administered drugs did not interfere with the assay. The limit of quantitation was 1.5 microg/l and the inter-day and intra-day relative standard deviations were less than 10%. Olanzapine was shown to be stable in plasma for up to 7 days when stored at 4 degrees C. Moreover, the addition of ascorbic acid was not necessary for the achievement of chemical stability during storage, or during the assay procedure. The method has been used to measure olanzapine concentrations in patients treated with various doses of the drug varying from 5 to 40 mg/day.     

不同提取溶剂和方法组合从落叶松毛虫蛹中提取的脂肪酸成分和含量的比较

落叶松毛虫Dendrolimus superans (Butler)蛹个体较大, 具有很高利用价值。为明确东北落叶松毛虫蛹中脂肪酸成分, 探讨最佳提取溶剂和提取方法的组合, 分别以正己烷、 石油醚和乙醚为提取溶剂, 结合超声波振荡萃取法、 索氏萃取法及溶剂萃取方法热浸和冷浸4种提取方法提取落叶松毛虫蛹油, 并采用毛细管色谱-质谱法分析提取物的脂肪酸种类和相对含量。结果表明： 正己烷溶剂与4种提取方法的组合中, 溶剂萃取热浸法提取率最高, 为25.60%。索氏萃取及溶剂萃取方法热浸和冷浸均检测到10种脂肪酸, 正己烷-超声波振荡萃取组合检测到9种脂肪酸。石油醚溶剂与4种提取方法的组合中, 索氏萃取提取率最高, 为29.31%, 均检测到10种脂肪酸。乙醚溶剂与4种提取方法的组合中, 溶剂萃取冷浸法提取率最高, 为29.11%, 检测到的脂肪酸种类为溶剂萃取冷浸法（13种）＞索氏萃取法（12种）＞溶剂萃取热浸法（11种）＞超声波振荡萃取法（9种）。在检测到的总脂肪酸中, 63%以上为不饱和脂肪酸, 其含量受提取溶剂和方法的影响不大。因此, 适合东北落叶松毛虫蛹中脂肪酸提取的最佳组合为石油醚溶剂 索氏萃取法。     

Gas chromatographic determination of bromo and fluoro derivatives of benzodiazepine in human body fluids

A rapid, sensitive and accurate method for the determination of bromazepam and flunitrazepam in plasma and urine using gas chromatography has been developed. Bromazepam was extracted with diethyl ether and flunitrazepam with hexane at pH 7. A nitrogen detector was used to determine bromazepam and an electron-capture detector was used for flunitrazepam.     

Toxicity of organic solvents used in situ in fermentation of lactic acid by Rhizopus arrhizus

Of 17 organic phases, some containing one of the five extractants for solvent extraction of lactic acid from Rhizopus arrhizus broth, solvents containing tertiary amines (Hostarex A327, trioctylamine) and secondary amine with isotridecanol modifier and trihexylphosphate, are from point of view of toxicity suitable for the in situ or on-line extraction. Pentaphosphinedipentylester is highly toxic. Toxicity of alcohols used as modifiers, decreases with an increase in the number of carbons in a molecule. Isotridecanol is medium toxic. Octanol and isodecanol and solvents containing them are toxic.This revised version was published online in October 2005 with corrections to the Cover Date.     

Fecal mutagens from subjects consuming a mixed-western diet

Because of potential significance of fecal mutagens (presumptive carcinogens) in the pathogenesis of colon cancer, feces from 99 healthy subjects from the New York metropolitan area were studied. The diet histories indicate that all participants were consuming a mixed-western diet which is high in total fat and low in fiber. Fecal samples that were incubated under anaerobic conditions at 37 degrees C for 96 h or frozen without incubation, were extracted with hexane: peroxide-free diethyl ether (1:1), partially purified on a silica Sep-pak cartridge and assayed for mutagenicity using the Salmonella typhimurium/mammalian microsome system. Aliquots of fecal samples incubated anaerobically showed a higher frequency of mutagenic activity (per cent samples showing activity) in strains TA98 and TA100 with and without microsomal (S9) activation. In addition, the mutagens requiring S9 activation, were more frequently inactivated when the fecal samples were frozen immediately after defecation and transported to the laboratory. Compared with hexane: ether, extraction of fecal samples with acetone increased the mutagenic activity mostly with TA98 with S9 activation. The HPLC fractionation of hexane: ether extract with methanol: water gradient using reverse phase C-18 column and UV detector at 254 nm indicated that the mutagenic activity (TA98 with S9 activation) is concentrated in several peaks. This is the first demonstration of HPLC profile of fecal samples that are active in TA98 with S9 activation. HPLC profile of fecal extracts and mutagenic activity of these extracts in strains TA98 and TA100 suggest the presence of several types of mutagens in the feces of healthy subjects consuming a high-fat, low-fiber mixed-western diet.     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11–0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasma with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 × 4.6 mm I.D., 5-μm column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than ±15%.     

Rapid high-performance liquid chromatographic determination of docetaxel (Taxotere) in plasma using liquid–liquid extraction

A new rapid and sensitive high-performance liquid chromatographic method for analysis of docetaxel (Taxotere) in human plasma was developed and validated. After adding an internal standard (paclitaxel, Taxol), plasma was extracted following a simple liquid–liquid extraction with diethyl ether. Extraction efficiency averaged 95% for docetaxel. Separation was performed using a Nucleosil (C₁₈) 5 μm column, monitored at 227 nm. The isocratic mobile phase consisted of acetonitrile–acetate buffer, pH 5–tetrahydrofuran (45:50:5, v/v) pumped at a flow-rate of 1.8 ml/min. The limit of quantification for docetaxel in plasma was 12.5 ng/ml. Retention times for docetaxel and paclitaxel were 7.7 and 9 min, respectively. Standard curves were linear over a range of 25–1000 ng/ml. This new method is rapid since it does not require time-consuming extraction procedures, or complex chromatographic conditions. This rapidity, along with the lack of chromatographic interferences with various other drugs likely to be administered to the cancer patients (pain killers, corticoids, antiemetics drugs) make this method suitable for daily routine analysis of Taxotere, a major anticancer drug extensively used in clinical oncology.     

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.