Identification and quantification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man

Identification of 6 beta-hydroxydexamethasone as a major urinary metabolite of dexamethasone in man has been accomplished by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Mass fragmentographic measurements revealed that more than 30% of the intravenously or orally administered dexamethasone dose was excreted in the 24-h urine as 6 beta-hydroxydexamethasone, while only a small fraction of the dose was excreted as unchanged dexamethasone and its glucuronic acid conjugate.     

Direct stereoselective assay of fluoxetine and norfluoxetine enantiomers in human plasma or serum by two-dimensional gas-liquid chromatography with nitrogen-phosphorus selective detection

A method was developed and validated for the direct enantioselective assay of fluoxetine and norfluoxetine in human plasma or serum by two-dimensional capillary gas-liquid chromatography (GC). A Rtx-1 fused-silica capillary (15 mx0.25 mm I.D., 1.0 micrometer film thickness) and a hydrodex-beta-6-TBDM fused-silica capillary (25 mx0.25 mm I.D., 0.25 micrometer film thickness) were used. A three-step liquid-liquid extraction was used for sample preparation with fluvoxamine and nisoxetine as internal standards. The method provided linear calibration between about 5 and 250 ng/ml for (R)- and (S)-fluoxetine as well as 15 and 250 ng/ml for (R)- and (S)-norfluoxetine. The limits of detection were about 1.5 and 6 ng/ml, respectively. Intra-day precision (coefficient of variation) was estimated as being between 5.4 and 12.7% at plasma levels of 25, 100 and 200 ng/ml for the four enantiomers. Inter-day precision was between 5.3 and 9.1% at 100 ng/ml. The enantioselective separation of some racemic psychopharmaceuticals was tested with various cyclodextrin GC-capillaries. Advantages and disadvantages of direct enantioselective GC are discussed for the assay of racemic psychopharmaceuticals. Samples from a patient who was treated with racemic fluoxetine were measured. In agreement with literature, plasma levels of the (R)-enantiomers of fluoxetine and norfluoxetine were considerably decreased in comparison to the (S)-enantiomers.     

Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization

A protocol utilizing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) using a simplified trimethylsilyl (TMS) derivatization protocol was developed and validated for the determination of hydroxylated metabolites of 3-keto-4-ene steroids such as testosterone, progesterone and androstenedione. Hydroxylated metabolites catalyzed by human CYP1B1 were extracted with methylene chloride and derivatized with BSTFA-10% TMCS. To get an optimal derivatizing condition, the effect of various incubation times and temperatures was evaluated. When the incubation temperature and time in the presence of the TMS derivatizing agent were increased, the 3-keto group became derivatized with TMS to form a 3-TMS derivative. To minimize the formation of the TMS ether on the 3-keto group, a reaction condition of 56 degrees C for 10 min was used for the routine measurement of the steroids and their hydroxylated metabolite. Performance studies including linearity of calibration curves, extraction efficiency and precision were performed. Linearity of the calibration curves was satisfactory from 0.125 to 5 microM for most compounds except 21-hydroxyprogesterone and 16alpha-hydroxyandrostenedione which deviated from linearity at the lower concentrations. Mean percentage extraction recoveries were greater than 80% for all compounds. Most compounds showed good precisions with C.V.s of within-day precision of less than 5% and C.V.s of between-day precision of less than 10%. The selected ion chromatograms from the recombinant human CYP1B1 incubations with testosterone, progesterone and androstenedione showed evidence of 6beta-, 16alpha-, 2alpha-, and 15alpha-hydroxytestosterone, 6alpha- and 16alpha-hydroxyprogesterone and 6alpha- and 16alpha-hydroxyandrostenedione, respectively. There was no significant interference associated with Escherichia coli membrane extracts in detecting hydroxylated metabolites. This procedure provides a rapid and sensitive method for the evaluation of steroid hydroxylation by CYP isoenzymes.     

Determination of mirtazapine in human plasma by liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of mirtazapine in human plasma is presented. The method is based on a liquid-liquid extraction and reversed-phase chromatography with fluorimetric detection. The separation was performed on a Luna microm C(18)(2) 50 x 4.6 mm I.D. column using an isocratic elution. Zolpidem hemitartrate was used as the internal standard. The between-day precision expressed by relative standard deviation was less than 5% and inaccuracy does not exceed 6%. A low limit of quantitation (1.5 ng/ml) and a short time of analysis (4 min) makes this assay suitable for pharmacokinetic studies.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.     

Validated method for rapid inhibition screening of six cytochrome P450 enzymes by liquid chromatography-tandem mass spectrometry

In vitro drug interaction data can be used in guiding clinical interaction studies, or, the design of new candidates. To make such a claim, it must be assured that the in vitro data obtained is confident. To meet this need, a rapid liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been validated and employed for routine screening of new chemical entities for inhibition of six major human cytochrome P450 (CYP) isoforms using cDNA-expressed CYPs. Probe substrates were used near the Michaelis-Menten constant (K(m)) concentration values for CYP1A2 (phenacetin), CYP2C9 (tolbutamide), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam and dextromethorphan). The major metabolites of CYP-specific probe substrates were quantified. The LC/MS/MS method was found to be accurate and precise within the linear range of 1.0-2000 ng/ml for each analyte in enzyme incubation mixture. The lower limit of quantification (LLOQ) was 1.0 ng/ml. The limit of detection (LOD) for the tested analytes was 0.48 ng/ml or better based on signal-to-noise ratio >3. The inhibition potential of the six CYP isoforms has been evaluated using their known selective inhibitors. The 50% inhibitory concentrations (IC(50) values) measured by this method demonstrated high precision and are consistent with the literature values.     

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

A sensitive and selective liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of novel trioxane antimalarials in different biomatrices using sample-pooling approach for high throughput pharmacokinetic studies

In the present studies, to give momentum to traditionally low throughput pharmacokinetic screening, a bioanalytical method based on the concept of sample pooling for simultaneous bioanalysis of multiple compounds is discussed. A sensitive, selective, specific and rapid HPLC/ESI-MS/MS assay method was developed and validated for the simultaneous quantitation of three novel trioxane antimalarials (99-357, 99-408 and 99-411) in rat plasma using trioxane analogue as internal standard. The suitably validated bioanalytical method was then further extrapolated to rabbit and monkey plasma by performing partial validation. Extraction from the plasma involves a simple two-step liquid-liquid extraction with n-hexane. The analytes were chromatographed on a cyano column by isocratic elution with acetonitrile:ammonium acetate buffer (pH 6) (85:15, v/v) and analyzed by mass spectrometry in multiple reaction-monitoring (MRM) mode. The chromatographic run time was 5.5 min and the weighted (1/x(2)) calibration curves were linear over a range of 1.56-200 ng/ml. The limit of detection (LOD) and lower limit of quantification (LLOQ) in rat plasma, rabbit plasma and monkey plasma were 0.78 and 1.56 ng/ml, respectively, for all three analytes. The intra- and inter-batch accuracy and precision in terms of % bias and % relative standard deviation were found to be well within the acceptable limits (< 15%). The average absolute recoveries of 99-357, 99-408 and 99-411 from spiked plasma samples were > 90%, > 70% and > 60%, respectively. The assay method described here could be applied to study the pharmacokinetics of 99-357, 99-408 and 99-411 using sample-pooling technique.     

Determination of the glucocorticoids prednisone, prednisolone, dexamethasone, and cortisol in human serum using liquid chromatography coupled to tandem mass spectrometry

Glucocorticoids are an important component of immunosuppressive therapy for solid organ transplantation. A method to quantitate prednisone, prednisolone, dexamethasone and cortisol in human serum has been developed. Analysis is performed utilizing reversed-phase liquid chromatography coupled to tandem mass spectrometry. The method was validated to a lower limit of quantitation of 5.4 ng/ml for prednisone and cortisol, and 10.7 ng/ml for dexamethasone and prednisolone, with error below 7% at the lower limits. The between-day relative standard deviations ranged 2.9-7.1%. Comparison of cortisol analysis to an established method using clinical samples yielded differences below 15% for 26 of 28 determinations.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

Gas chromatographic determination of midazolam in low-volume plasma samples

A gas chromatographic method for the sensitive determination of midazolam in plasma volumes as low as 40 μl was developed, utilizing clinazolam as the internal standard. After liquid-liquid extraction at basic pH into 1-chlorobutane-dichloromethane (96:4) a 2- to 4-μl portion of the reconstituted extract was injected under electronic pressure control onto a 12 m × 0.2 mm I.D. methyl silicone capillary column, and was exposed to a three-step temperature program from 120 to 310°C, to separate the analytes from the plasma constituents. The compound of interest was identified and quantified by means of a mass-selective detector. The assay was linear from 10 to 500 ng/ml using 40 μl of plasma (limit of quantification: 10 ng/ml) and was linear from 0.25 to 100 ng/ml using 500 μl of plasma (limit of quantification: 0.25 ng/ml). The intra-day precision for the 40-μl aliquots varied from 2.2 to 6.6%, the corresponding accuracy from −7.4 to −4.4%; the inter-day precision ranged from 5 to 7.2% and the corresponding accuracy from −7.2 to −5.1%.     

A highly sensitive LC-MS-MS assay for analysis of midazolam and its major metabolite in human plasma: applications to drug metabolism

Betamethasone is a synthetic corticosteroid designed to exert a marked glucocorticoid activity. As the free alcohol, betamethasone finds widespread clinical applications related to its anti-inflammatory and immunosuppressant activity. In the present study, a fast, sensitive, robust method was developed for the determination and quantification of betamethasone in human plasma by liquid chromatography coupled with tandem mass spectrometry, using photospray ionization in negative mode. Betamethasone was extracted from 0.5 ml human plasma by liquid-liquid extraction (LLE) using chloramphenicol as internal standard. The method has a chromatographic run of 2.5 min using a C(18) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 0.05 to 50 ng ml(-1) (r(2)>0.993). The between-run precision, based on the relative standard deviation replicate quality controls was 94.1% (0.15 ng ml(-1)), 90.7% (4.0 ng ml(-1)) and 97.2% (40 ng ml(-1)). The between-run accuracy for the above-mentioned concentrations was 11.9, 9.0 and 9.8%, respectively. The method herein described was employed in a bioequivalence study of two formulations of dexchlorpheniramine/betamethasone 2 mg/0.25 mg tablets.     

Liquid chromatographic-mass spectrometric method for the determination of alpha-,beta-arteether in rat serum

This study reports the development and validation of a sensitive and selective assay method for the determination of alpha-,beta-arteether in rat serum by liquid chromatography-mass spectrometry. The mobile phase was composed of methanol-0.1 mM sodium acetate (pH 5) (80:20%) at a flow-rate of 1 ml min(-1) and chromatographic separations were achieved on a Ultracarb, 5 ODS 20, Phenomenex column (5 micrometer, 30 mmx4.6 mm I.D.). The total effluent from the column was split so that one-tenth was injected into the electrospray LC-MS interface. ESI-MS analysis was carried out using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at a cone voltage of 52 V with a scan range of 100-400 Da. The analytes were quantified from the [M+Na](+) ion chromatograms of alpha-,beta-arteether at m/z 335 and artemisinin at m/z 305. A simple liquid-liquid extraction with 2x2 ml n-hexane was used to isolate alpha-,beta-arteether from rat serum. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recovery from spiked control samples ranged from 88.41 to 96.17% with a maximum CV of 10.8% for alpha-arteether and 69.83-79.69% with a maximum CV of 17.06% for beta-arteether. Linearity in serum was observed over the range 20-320 ng ml(-1). Percent bias (accuracy) was well within the acceptable range. Within- and between-assay precision were less than 15%. The assay method described here is being applied to study the pharmacokinetics of CDRI developed intramuscular formulation Emal (alpha-/beta-arteether in the ratio of 30:70) in rats. The method is sensitive enough to monitor alpha-,beta-arteether up to 24 h after a single 30 mg kg(-1) i.m. dose.     

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile-50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm x 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9-108.3% for CPT-11 in culture media and 94.3-107.2% for CPT-11 in cell lysates; and 87.7-106.8% for SN-38 in culture media and 90.1-105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies

A high-performance liquid chromatography (LC-MS) method has been developed and validated for the determination of dexamethasone in dried blood spot (DBS) samples. For the preparation of DBS samples whole blood spiked with analyte was used to produce 30μl blood spots on specimen collection cards. An 8mm disc was cut from the DBS sample and extracted using a combination of methanol: water (70:30, v/v) containing the internal standard, triamcinolone acetonide. Extracts were centrifuged and chromatographic separation was achieved using a Zorbax Eclipse Plus C18 column using gradient elution with a mobile phase of acetonitrile and water with formic acid at a flow rate of 0.2ml/min. LC-MS detection was conducted with single ion monitoring using target ions at m/z 393.1 for dexamethasone and 435.1 for the internal standard. The developed method was linear within the tested calibration range of 15-800ng/ml. The overall extraction recovery of dexamethasone from DBS samples was 99.3% (94.3-105.7%). The accuracy (relative error) and precision (coefficient of variation) values were within the pre-defined limits of ≤15% at all concentrations. Factors with potential to affect drug quantification measurements such as blood haematocrit, the volume of blood applied onto the collection card and spotting device were investigated. Although a haematocrit related effect was apparent, the assay accuracy and precision values remained within the 15% variability limit with fluctuations in haematocrit of ±5%. Variations in the volume of blood spotted did not appear to affect the performance of the developed assay. Similar observations were made regarding the spotting device used. The methodology has been applied to determine levels of dexamethasone in DBS samples collected from premature neonates. The measured concentrations were successfully evaluated using a simple 1-compartment pharmacokinetic model. Requiring only a microvolume (30μl) blood sample for analysis, the developed assay is particularly suited to pharmacokinetic studies involving paediatric populations.     

Dexamethasone suppressibility of plasma dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one) in normal men

9 AM and overnight dexamethasone suppression tests were performed in normal adult subjects and plasma dehydroepiandrosterone (DHA) levels were radioimmunoassayed. The results were as follows : 1) In the 9 AM test, plasma DHA was suppressed to the lowest level at the time between 4 hours and 6 hours after dexamethasone; 2) 2 mg (overnight test) or 3 mg (9AM test) of dexamethasone induced the maximum DHA suppression; 3) after dexamethasone administration in both the tests, plasma DHA was not suppressed below 30 % of the basal level, nor below 2 ng/ml; and 4) there was no significant difference in dexamethasone suppressibility of plasma DHA between 9 AM test and overnight test.     

Liquid chromatography-positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma

A rapid, sensitive and novel narrow-bore liquid chromatography-mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid-liquid extraction with a mixture of hexane-heptane-isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C(8) micro-bore column (250 mm x 2.1 mm i.d.; 3.5 microm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate-formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min(-1). The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50-250 ng ml(-1) (r(2) > 0.996). The limit of quantitation was 0.50 ng ml(-1). Accuracy and precision were below the acceptance limits of 15%.     

Simultaneous determination of cyclandelate and its metabolite in human plasma by capillary column gas-liquid chromatography

A method was developed for the simultaneous determination of cyclandelate and mandelic acid concentrations in plasma, involving extraction from plasma followed by trimethylsilylation and chromatography of the derivatives on a glass capillary column with hydrogen flame-ionization detection. Calibration graphs were linear down to at least 20 μg/ml for each substance. The precision was excellent with a pooled relative standard deviation of 6.3% and 6.4% for cyclandelate and mandelic acid serum samples, respectively. Concentrations below 500 ng/ml of each substance could be detected in human plasma. The method was developed for use in bioavailability and metabolism studies.