Characterizing the impact of minor cannula design modification.

OBJECTIVE: Bench evaluation of the hydrodynamic behavior of venous cannulas is a valuable technique for the analysis of their performance during cardiopulmonary bypass (CPB). The aim of this study was to investigate the effect of the internal diameter of the extracorporeal connecting tube of venous cannulas on flow rate (Q), pressure drop (delta P), and cannula resistance (delta P/Q²) values, using a computer assisted test bench.¦METHODS: An in vitro circuit was set up with silicone tubing between the test cannula encased in a movable reservoir, and a static reservoir. The delta P, defined as the difference between the drainage pressure and the preload pressure, was measured using high-fidelity Millar pressure transducers. Q was measured using an ultrasonic flowmeter. Data display and data recording were controlled using virtual instruments in a stepwise fashion.¦RESULTS: The 27 F smartcanula® with a 9 mm connecting tube diameter showed 17% less resistance compared to that with an 8 mm connecting tube diameter. Q values were 7.22±0.1 and 7.81±0.04 L/min for cannulas with 8 mm and 9 mm connecting tube diameters, respectively. The delta P/Q² ratio values were 72% lower for the Medtronic cannula with a 9 mm connecting tube diameter compared to that with an 8 mm connecting tube diameter. Q values for the Medtronic cannula were 3.94±0.23 and 6.58±0.04 L/min with 8 mm and 9 mm connecting tube diameters, respectively. The 27 F smartcanula® showed 13% more flow rate compared to the 28 F Medtronic cannula using the unpaired Student t-test (p<0.0001).¦CONCLUSIONS: Our results demonstrated that Q was increased but delta P and delta P/Q² values were significantly decreased when the connecting tube diameter was increased for venous cannulas. The connecting tube diameter significantly affected the resistance to liquid flow through the cannula. Smartcanulas® outperform Medtronic cannulas.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

A phase I pharmacokinetic study of hypoxic abdominal stop-flow perfusion with gemcitabine in patients with advanced pancreatic cancer and refractory malignant ascites

PURPOSE: As no curative treatment for advanced pancreatic and biliary cancer with malignant ascites exists, new modalities possibly improving the response to available chemotherapies must be explored. This phase I study assesses the feasibility, tolerability and pharmacokinetics of a regional treatment of gemcitabine administered in escalating doses by the stop-flow approach to patients with advanced abdominal malignancies (adenocarcinoma of the pancreas, n = 8, and cholangiocarcinoma of the liver, n = 1). EXPERIMENTAL DESIGN: Gemcitabine at 500, 750 and 1,125 mg/m(2) was administered to three patients at each dose level by loco-regional chemotherapy, using hypoxic abdominal stop-flow perfusion. This was achieved by an aorto-caval occlusion by balloon catheters connected to an extracorporeal circuit. Gemcitabine and its main metabolite 2',2'-difluorodeoxyuridine (dFdU) concentrations were measured by high performance liquid chromatography with UV detection in the extracorporeal circuit during the 20 min of stop-flow perfusion, and in peripheral plasma for 420 min. Blood gases were monitored during the stop-flow perfusion and hypoxia was considered stringent if two of the following endpoints were met: pH </= 7.2, pO(2) nadir ratio </=0.70 or pCO(2) peak ratio >/=1.35. The tolerability of this procedure was also assessed. RESULTS: Stringent hypoxia was achieved in four patients. Very high levels of gemcitabine were rapidly reached in the extracorporeal circuit during the 20 min of stop-flow perfusion, with C (max) levels in the abdominal circuit of 246 (+/-37%), 2,039 (+/-77%) and 4,780 (+/-7.3%) mug/ml for the three dose levels 500, 750 and 1,125 mg/m(2), respectively. These C (max) were between 13 (+/-51%) and 290 (+/-12%) times higher than those measured in the peripheral plasma. Similarly, the abdominal exposure to gemcitabine, calculated as AUC(t0-20), was between 5.5 (+/-43%) and 200 (+/-66%)-fold higher than the systemic exposure. Loco-regional exposure to gemcitabine was statistically higher in presence of stringent hypoxia (P < 0.01 for C (max) and AUC(t0-20), both normalised to the gemcitabine dose). Toxicities were acceptable considering the complexity of the procedure and were mostly hepatic; it was not possible to differentiate the respective contributions of systemic and regional exposures. A significant correlation (P < 0.05) was found between systemic C (max) of gemcitabine and the nadir of both leucocytes and neutrophils. CONCLUSIONS: Regional exposure to gemcitabine-the current standard drug for advanced adenocarcinoma of the pancreas-can be markedly enhanced using an optimised hypoxic stop-flow perfusion technique, with acceptable toxicities up to a dose of 1,125 mg/m(2). However, the activity of gemcitabine under hypoxic conditions is not as firmly established as that of other drugs such as mitomycin C, melphalan or tirapazamine. Further studies of this investigational modality, but with bioreductive drugs, are therefore warranted first to evaluate the tolerance in a phase I study and later on to assess whether it does improve the response to chemotherapy.

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

The Effect of Adding CO2 to Hypoxic Inspired Gas on Cerebral Blood Flow Velocity and Breathing during Incremental Exercise

Hypoxia increases the ventilatory response to exercise, which leads to hyperventilation-induced hypocapnia and subsequent reduction in cerebral blood flow (CBF). We studied the effects of adding CO2 to a hypoxic inspired gas on CBF during heavy exercise in an altitude naïve population. We hypothesized that augmented inspired CO2 and hypoxia would exert synergistic effects on increasing CBF during exercise, which would improve exercise capacity compared to hypocapnic hypoxia. We also examined the responsiveness of CO2 and O2 chemoreception on the regulation ventilation (E) during incremental exercise. We measured middle cerebral artery velocity (MCAv; index of CBF), E, end-tidal PCO2, respiratory compensation threshold (RC) and ventilatory response to exercise (E slope) in ten healthy men during incremental cycling to exhaustion in normoxia and hypoxia (FIO2 = 0.10) with and without augmenting the fraction of inspired CO2 (FICO2). During exercise in normoxia, augmenting FICO2 elevated MCAv throughout exercise and lowered both RC onset andE slope below RC (P<0.05). In hypoxia, MCAv and E slope below RC during exercise were elevated, while the onset of RC occurred at lower exercise intensity (P<0.05). Augmenting FICO2 in hypoxia increased E at RC (P<0.05) but no difference was observed in RC onset, MCAv, or E slope below RC (P>0.05). The E slope above RC was unchanged with either hypoxia or augmented FICO2 (P>0.05). We found augmenting FICO2 increased CBF during sub-maximal exercise in normoxia, but not in hypoxia, indicating that the 'normal' cerebrovascular response to hypercapnia is blunted during exercise in hypoxia, possibly due to an exhaustion of cerebral vasodilatory reserve. This finding may explain the lack of improvement of exercise capacity in hypoxia with augmented CO2. Our data further indicate that, during exercise below RC, chemoreception is responsive, while above RC the ventilatory response to CO2 is blunted.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Cytostatic lung perfusion results in heterogeneous spatial regional blood flow and drug distribution: evaluation of different cytostatic lung perfusion techniques in a porcine model.

OBJECTIVES: Comparison of doxorubicin uptake, leakage and spatial regional blood flow, and drug distribution was made for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), as opposed to intravenous administration in a porcine model. METHODS: White pigs underwent single-pass lung perfusion with doxorubicin (320 mug/mL), labeled 99mTc-microspheres, and Indian ink. Visual assessment of the ink distribution and perfusion scintigraphy of the perfused lung was performed. 99mTc activity and doxorubicin levels were measured by gamma counting and high-performance liquid chromatography on 15 tissue samples from each perfused lung at predetermined localizations. RESULTS: Overall doxorubicin uptake in the perfused lung was significantly higher (P = .001) and the plasma concentration was significantly lower (P < .0001) after all isolated lung perfusion techniques, compared with intravenous administration, without differences between them. Pulmonary artery infusion (blood flow occlusion) showed an equally high doxorubicin uptake in the perfused lung but a higher systemic leakage than surgical isolated lung perfusion (P < .0001). The geometric coefficients of variation of the doxorubicin lung tissue levels were 175%, 279%, 226%, and 151% for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), respectively, compared with 51% for intravenous administration (P = .09). 99mTc activity measurements of the samples paralleled the doxorubicin level measurements, indicating a trend to a more heterogeneous spatial regional blood flow and drug distribution after isolated lung perfusion and blood flow occlusion compared with intravenous administration. CONCLUSIONS: Cytostatic lung perfusion results in a high overall doxorubicin uptake, which is, however, heterogeneously distributed within the perfused lung.

Antigenic heterogeneity of clones and subclones from human melanoma cell lines demonstrated by a panel of monoclonal antibodies and flow microfluorometry analysis.

Cells from two melanoma cell lines, Me43 and GLL-19, were cloned in methylcellulose cultures and 20 randomly selected colonies from each line were picked up by micromanipulation, expanded in liquid cultures, and considered as clones of the original cell lines. The antigenic cell surface phenotype of these clones defined by panel of 12 monoclonal antibodies (MAb) was analyzed by flow microfluorometry (FMF) using a fluorescence-activated cell sorter (FACS II) and compared with the known stable phenotype of the parent cell line. The antibody panel consisted of eight MAb against melanoma-associated antigens, two MAb against monomorphic determinants of HLA-DR (la) and HLA-ABC, respectively, one MAb against the common acute lymphoblastic leukemia antigen (CALLA) and one MAb against carcinoembryonic antigen used as control. A remarkable heterogeneity in terms of qualitative and quantitative expression of the cell surface antigens studied was observed among and within the different clones. The single-cell origin of the clones was assessed by comparing the clonogenic cell frequency, determined by limiting dilutions in microculture plates, with the cloning efficiency observed in Petri dishes. Both techniques using methylcellulose medium gave the same percentages of growing colonies. Cells from four Me43 clones were recloned in methylcellulose and the phenotype of five randomly selected subclones from each clone was analysed using the same panel of monoclonal antibodies. Each subclone also displayed heterogeneity with individual phenotypes different from that of the original clone and from the parental Me43 cell line. The antigen expression by individual cells in situ within clones was analyzed on frozen sections from colonies using the same panel of MAb and a biotin-avidin immunoperoxidase method. The results confirmed the marked heterogeneity of antigen expression within and among colonies, as indicated by the FMF analysis.

Loco-regional administration of anticancer agents : isolated lung perfusion with doxorubicin hypoxic abdominal stop-flow perfusion with gemcitabine

SUMMARY Regional drug delivery is an approach designed to improve the selectivity of anticancer chemotherapy. The advantage of regional treatments lies in increasing the drug concentration in the affected organ, while the rest of the organism is spared, thus improving efficacy and limiting treatment toxicity. The goal of this thesis was to assess the distribution throughout the body and the disposition (pharmacokinetics) of two anticancer agents, doxorubicin and gemcitabine, administered by two different regional administration modalities: isolated lung perfusion (ILP) for pulmonary metastases from soft tissue sarcomas and abdominal stop-flow hypoxic perfusion for advanced pancreatic cancers, respectively. For this purpose, two high-performance liquid chromatography methods were developed and validated. The first enabled the determination of doxorubicin in four different biological matrices: serum, reconstituted effluent, tissues with low levels of doxorubicin and tissues with high levels of doxorubicin. The second allows the analysis of gemcitabine and its principal metabolite dFdU in plasma. The administration of doxorubicin by ILP was studied in three preclinical studies (one on pigs and two on rats). It was first shown that, regardless of the administration mode, doxorubicin was not homogeneously distributed throughout the lung and that some regions remained out of reach. Secondly, it was demonstrated that doxorubicin did not adequately reach the tumours despite very high levels found in the lung. Finally, an attempt to enhance the doxorubicin tumoural uptake by pharmacologic modulation using two P-glycoprotein inhibitors, cyclosporin and valspodar, was unsuccessful. The last part of this work involves the administration of gemcitabine by abdominal stop-flow as a part of a phase I clinical trial in patients with advanced pancreatic disease or resistant malignant ascites. The study has demonstrated that the regional exposure to gemcitabine was increased while the exposure of the entire organism was similar to standard intravenous administrations. From a toxicological perspective, the procedure was rather well tolerated. However, even if no clinical response is expected from a phase I study, no hints of clinical responses were unfortunately observed. In conclusion, even if loco-regional therapies may afford the pharmacological advantage of increasing anticancer drug levels at the tumour site, further studies of these investigational treatment modalities are warranted to ascertain whether they can provide a significant improvement of the cancer therapy for patients, in terms of treatment tolerability, improved responses and survival rates. RÉSUMÉ L'administration locorégionale d'agents anticancéreux est une approche destinée à augmenter la sélectivité du traitement. L'avantage des traitements régionaux repose sur le fait que la concentration du médicament cytostatique est augmentée dans l'organe où est localisée la tumeur, alors que le reste de l'organisme est épargné, améliorant ainsi en théorie l'efficacité du traitement et en limitant sa toxicité. Le but de ce travail de thèse avait pour objectif de préciser, la pharmacocinétique au sein de l'organisme de deux agents anticancéreux, la doxorubicine et la gemcitabine, administrés par deux types de perfusions loco-régionales: la perfusion isolée du poumon (ILP) pour les métastases pulmonaires de sarcomes des tissus mous, et la perfusion hypoxique (stop-flow) abdominale pour les cancers avancés du pancréas. Dans cette optique, deux méthodes de chromatographie liquide à haute performance ont été développées et validées. La première permet le dosage de la doxorubicine dans quatre milieux biologiques: le sérum, l'effluent reconstitué, ainsi que des tissus contenant des concentrations faibles et élevées en doxorubicine. La seconde méthode permet le dosage dans le plasma de la gemcitabine et de son principal métabolite, le dFdU. L'administration de doxorubicine par ILP a été étudiée dans trois études précliniques (une chez le porc et deux chez le rat). Il a été montré, dans un premier temps, que la doxorubicine n'était pas distribuée de façon homogène au sein du poumon, quel que soit son mode d'administration. Dans un deuxième temps, il a été démontré que le médicament n'atteignait pas les tumeurs de façon adéquate, malgré des concentrations très élevées au sein du tissu pulmonaire. Finalement, une tentative d'augmenter la pénétration tumorale de la doxorubicine par une modulation pharmacologique de la P-glycoprotéine en utilisant la cyclosporine et le valspodar n'a pas abouti. La dernière partie de ce travail concernait l'administration de gemcitabine par stop-flow abdominal dans le cadre d'une étude clinique de phase I menée auprès de patients atteints de cancers avancés du pancréas ou d'ascites malignes réfractaires. Cette étude a démontré que l'exposition régionale à la gemcitabine était augmentée, alors que l'exposition de l'organisme était similaire à une administration de dose standard par voie intraveineuse. D'un point de vue toxicologique la procédure fut relativement bien tolérée. Cependant, même s'il n'est pas attendu de réponses cliniques dans une étude de phase I, aucun signe de réponse au traitement n'a pu être malheureusement observé. En conclusion, même si les thérapies loco-régionales présentent -en théorie- l'avantage pharmacologique d'augmenter les taux du médicaments anticancéreux sur le site de la tumeur, d'autres études précliniques et cliniques sont nécessaires pour démontrer que ces nouvelles modalités de traitement, de nature investigationelle à présent, apportent une réelle amélioration pour la prise en charge des patients cancéreux, en terme de tolérance au traitement et de l'augmentation des taux de réponses et de survie.

Response-to-comments about: "Is it really the method for carbon dioxide determination in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry valid?" from T. Saffaj and B. Ihssane

Saffaj et al. recently criticized our method of monitoring carbon dioxide in human postmortem cardiac gas samples using Headspace-Gas Chromatography-Mass Spectrometry. According to the authors, their demonstration, based on the latest SFSTP guidelines (established after 2007 [1,2]) fitted for the validation of drug monitoring bioanalytical methods, has put in evidence potential errors. However, our validation approach was built using SFSTP guidelines established before 2007 [3-6]. We justify the use of these guidelines because of the post-mortem context of the study (and not clinical) and the gaseous state of the sample (and not solid or liquid). Using these guidelines, our validation remains correct.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Tandem use of solid-phase extraction and dispersive liquid-liquid microextraction for the determination of mononitrotoluenes in aquatic environment.

Solid-phase extraction (SPE) in tandem with dispersive liquid-liquid microextraction (DLLME) has been developed for the determination of mononitrotoluenes (MNTs) in several aquatic samples using gas chromatography-flame ionization (GC-FID) detection system. In the hyphenated SPE-DLLME, initially MNTs were extracted from a large volume of aqueous samples (100 mL) into a 500-mg octadecyl silane (C(18) ) sorbent. After the elution of analytes from the sorbent with acetonitrile, the obtained solution was put under the DLLME procedure, so that the extra preconcentration factors could be achieved. The parameters influencing the extraction efficiency such as breakthrough volume, type and volume of the elution solvent (disperser solvent) and extracting solvent, as well as the salt addition, were studied and optimized. The calibration curves were linear in the range of 0.5-500 μg/L and the limit of detection for all analytes was found to be 0.2 μg/L. The relative standard deviations (for 0.75 μg/L of MNTs) without internal standard varied from 2.0 to 6.4% (n=5). The relative recoveries of the well, river and sea water samples, spiked at the concentration level of 0.75 μg/L of the analytes, were in the range of 85-118%.

Determination of plasma levels of citalopram and its demethylated and deaminated metabolites by gas chromatography and gas chromatography-mass spectrometry.

Sensitive and specific methods based on gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) for the determination of levels of citalopram, desmethylcitalopram and didesmethylcitalopram in the plasma of patients treated with citalopram are presented, as well as a GC-MS procedure for the assay of the citalopram propionic acid derivative. After addition of a separate internal standard for each drug, liquid-solvent extraction is used to separate the basic compounds from the acid compounds. The demethylated amines are derivatized with trifluoroacetic anhydride, and the acid metabolite with methyl iodide. GC-MS is performed in the electron impact mode, as mass spectrometry by the (positive-ion) chemical ionization mode (methane and ammonia) appeared to be unsuitable. The limits of quantification were 1 ng/ml for citalopram and desmethylcitalopram and 2 ng/ml for the other metabolites. The correlation coefficients for the calibration curves (range 10-500 ng/ml) were &gt; or = 0.999 for all compounds, whether determined by GC or GC-MS.