Quality Control Concentrations Research Articles

Simultaneous quantification of beclomethasone dipropionate and its metabolite, beclomethasone 17-monopropionate in rat and human plasma and different rat tissues by liquid chromatography–positive electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography–positive electrospray ionization tandem mass spectrometry (LC–(ESI+)-MS–MS) method has been developed and validated for the simultaneous quantification of beclomethasone dipropionate (BDP) and its active metabolite, beclomethasone 17-monopropionate (17-BMP) in rat plasma and different tissues using fluticasone propionate (FP) as the internal standard. The method was validated over a linear range from 0.05 to 5 ng/ml for both analytes. A solid-phase extraction procedure was used for plasma samples and a liquid–liquid extraction procedure for tissues samples (lung, liver and kidney). The between-day and within-day coefficients of variation for all compounds were ≤20% at the concentrations of lower limit of quantification (LLOQ) and ≤15% at other quality control concentrations. The same method was also partially validated for human plasma. The utility of this assay was demonstrated by monitoring BDP and 17-BMP plasma and tissue concentrations in an animal study designed for evaluation of pulmonary targeting after intratracheal administration of BDP dry powder.

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile–methanol–0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 μl) of the supernatant was injected into the HPLC system linked to a Waters XTerra™ RP 18 column ( 150 mm×4.6 mm i.d., particle size 5 μm). The compound was eluted by a mixture of acetonitrile–methanol–0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50–1200 ng/ml and 1200–12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from −2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration–time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.

Quantitative determination of DRF-1042 in human plasma by HPLC: validation and application in clinical pharmacokinetics.

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of DRF-1042, a novel orally active camptothecin (CPT) analog, in human plasma. The sample preparation was a simple deproteinization with acidified methanol yielding almost 100% recovery of DRF-1042. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 x 4.6 mm, 5 microm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a fl ow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. The standard curves were linear (r(2) > 0.999) in the concentration ranges 5.0-2004 ng/mL. The lower limit of quantification (LLQ) of the assay was 5 ng/mL. The mean measured quality control (QC) concentrations (range 5 ng/mL to 40 microg/mL) deviated from the nominal concentrations in the range of -10.5-0.08 and -14.5-7.97%, inter- and intra-day, respectively. The inter- and intra-day precisions in the measurement of QC samples at four tested concentrations, were in the range 0.64-5.89% relative standard deviation (RSD) and 0.33-14.7% RSD, respectively. The method was found to be suitable for measurement of plasma concentrations above the calibration curve after serial dilutions. Stability of DRF-1042 was confirmed in a battery of studies, viz., on bench-top, in the auto-sampler, in the stock solutions, after four quick freeze-thaw cycles, up to one month at -20 degree C in human plasma and up to 2 months in the ex vivo samples. The method is simple, sensitive and reliable and has been successfully implemented to investigate the clinical pharmacokinetics of DRF-1042 in cancer patients in a phase I clinical trial.

Simultaneous quantification of budesonide and its two metabolites, 6β‐hydroxybudesonide and 16α‐hydroxyprednisolone, in human plasma by liquid chromatography negative electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography negative electrospray ionization tandem mass spectrometry [LC-(-)ESI-MS-MS] method has been developed and validated for the simultaneous quantification of budesonide (BUD) and its major metabolites, 6beta-hydroxybudesonide (OH-BUD) and 16alpha-hydroxyprednisolone (OH-PRED) in human plasma. The method was validated over a linear range from 0.1 to 10 ng/mL for all three analytes using a solid-phase extraction procedure with 9-fluoro-hydrocortisone as the internal standard. The between-day and within-day coefficients of variation for all compounds were < or =20% at the concentrations of lower limit of quantification and < or =15% at other quality control concentrations. The utility of this assay was demonstrated by monitoring BUD, OH-BUD and OH-PRED plasma concentrations in one healthy subject for 24 h following a 3 mg oral dose of budesonide, administered as a pH modified release capsule (Budenofalk) to healthy volunteers.

Quantification of the O- and N-demethylated metabolites of hydrocodone and oxycodone in human liver microsomes using liquid chromatography with ultraviolet absorbance detection

High-performance liquid chromatographic assays for the O- and N-demethylated oxidative metabolites of hydrocodone and oxycodone formed in human liver microsomes are described. A solvent–solvent extraction/re-extraction procedure followed by reversed-phase HPLC with UV detection at 210 nm allows for the quantification of hydromorphone, norhydrocodone, oxymorphone and noroxycodone. Calibration curve concentration ranges were 0.63–400 μ M (0.18–114 μg/ml) and 1.25–400 μ M (0.36–114 μg/ml) for hydromorphone and norhydrocodone, respectively and 0.13–20 μ M (0.04–6.03 μg/ml) and 1–200 μ M (0.30–60 μg/ml) for oxymorphone and noroxycodone, respectively. Assay performance was determined by intra- and inter-assay precision and inaccuracies for quality control samples and was <15% for all metabolites at each quality control concentration. These methods provide good precision, accuracy and sensitivity for use in in vitro kinetic studies investigating the oxidative metabolism of hydrocodone and oxycodone in human liver microsomes.

High performance liquid chromatographic-mass spectrometric assay for the quantitation of BMS-204352 in dog K(3)EDTA plasma.

A high performance liquid chromatographic-mass spectrometric (LC/MS) assay was developed and validated for the determination of BMS-204352 in dog K(3)EDTA plasma. A 0.5 mL aliquot of control plasma was spiked with BMS-204352 and internal standard (IS) and buffered with 1 mL of 5 mM ammonium acetate. The mixture was then extracted with 3 mL of toluene. After separation and evaporation of the organic phase to dryness using nitrogen at 40 degrees C, the residue was reconstituted in the mobile phase and 25 microL of the sample were injected onto a Hypersil C(18) column (2 x 50 mm; 3 microm) at a flow rate of 0.5 mL/min. The mobile phase was consisted of two solvent mixtures (A and B). Solvent A was composed of 5 mM ammonium acetate and 0.1% triethylamine in 75:25 v/v water:methanol, pH adjusted to 5.5 with glacial acetic acid, and solvent B was 5 mM ammonium acetate in methanol. A linear gradient system was used to elute the analytes. The mass spectrometer was programmed to admit the de-protonated molecules at m/z 352.7 (IS) and m/z 357.9 (BMS-204352). Standard curves of BMS-204352 were linear (r(2) > or = 0.998) over the concentration range of 0.5-1000 ng/mL. The mean predicted quality control (QC) concentrations deviated less than 5.1% from the corresponding nominal values (ie 4, 80, 400 and 2000 ng/mL); the within- and between-assay precision of the assay were within 5.5% relative standard deviation. Stability of BMS-204352 was confirmed after at least three freeze/thaw cycles and BMS-204532 was stable in dog plasma when stored frozen at or below -20 degrees C for at least 16 weeks in spiked QC samples and for at least 4 1/2 weeks for in vivo study samples. BMS-204352 and IS were stable in the injection solvent at room temperature for at least 24 h. The assay was applied to delineate the pharmacokinetic disposition of BMS-204352 in dogs following a single intravenous dose administration. In conclusion, the assay is accurate, precise, specific, sensitive and reproducible for the pharmacokinetic analysis of BMS-204532 in dog plasma.

Determination of unbound concentration of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in human plasma by ultrafiltration followed by high-performance liquid chromatography with fluorimetric detection

The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a highly protein bound drug with narrow therapeutic window. We report a simple HPLC method with fluorimetric detection for the determination of free DMXAA concentration in human plasma. Sample preparation involves the ultrafiltration of plasma by a Centrisart device for 30 min at 2000 g and extraction with acetonitrile: methanol mixture. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed at the concentration range of 0.5–40 μ M in blank plasma and phosphate buffer. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The HPLC method has been used for the analysis of preclinical studies.

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C 18 column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 m M ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μ M in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μ M. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 m M phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 m M sodium dihydrogen phosphate (pH 3.4), 10 m M acetic acid and 36μ M EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μ M and the calibration line was linear up to 75 μ M. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μ M) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μ M, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μ M, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.

Simultaneous quantification of tacrolimus and sirolimus, in human blood, by high-performance liquid chromatography-tandem mass spectrometry.

In this paper the authors present a validated method for the simultaneous analysis of tacrolimus and sirolimus in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry. Blood samples (500 microL) were prepared by C18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring. The assay was linear for both compounds over the range 0.25-100 microg/L (r2 > 0.996, n = 7). At the limit of quantification (0.25 microg/L), for both sirolimus and tacrolimus, the interday imprecision was < 3% and the analytical recovery was between 97.0% and 102%, respectively. The interbatch and intrabatch coefficients of variation of the method for both analytes, at the three quality control concentrations (0.5, 20, and 80 microg/L), were < 16% and < 10%, respectively. The analytical recovery, at the three control concentrations, ranged from 99.2% to 104% of the nominal concentration. The mean absolute recovery (+/- standard deviation) of tacrolimus, sirolimus, and internal standard was 82 +/- 7%, 89 +/- 12%, and 77 +/- 8%, respectively (n = 12). In conclusion, the method presented can be used for simultaneous determination of tacrolimus and sirolimus and will aid in pharmacokinetic studies and therapeutic drug monitoring of these drugs. Furthermore, this method has economic benefits in the clinical setting where these drugs are coadministered.

Simultaneous quantitation of d7‐nefazodone, nefazodone, d7‐hydroxynefazodone, hydroxynefazodone, m‐chlorophenylpiperazine and triazole‐dione in human plasma by liquid chromatographic‐mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 µL of supernatant was mixed with 100 µL of 10 mM ammonium formate (pH = 4.0), and a 50 µL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r2 ≥ 0.994) over the concentration range of 4–1000 ng/mL for Dione and 2–500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally. Copyright © 2000 John Wiley & Sons, Ltd.

Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in human plasma by liquid chromatographic-mass spectrometry.

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 microL of supernatant was mixed with 100 microL of 10 mM ammonium formate (pH = 4.0), and a 50 microL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) >/= 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally.

Determination of two major metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid in hepatic microsomal incubations by high-performance liquid chromatography with fluorescence detection

High-performance liquid chromatographic methods have been developed and validated for the glucuronidated and oxidative metabolites of the novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), produced in human liver microsomal incubations. Calibration curves for DMXAA acyl glucuronide (DMXAA-Glu) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) were constructed over the concentration ranges of 0.25 to 20 and 0.5 to 40 μ M, respectively. Assay performance was determined by intra- and inter-day accuracy and precision of quality control (QC) samples. The difference between the theoretical and measured concentration, and the coefficient of variation, were less than 15% at low QC concentrations, and less than 10% at medium and high QC concentrations for both analytes. The methods presented good accuracy, precision and sensitivity for use in kinetic studies of the glucuronidated and oxidative metabolites of DMXAA in human liver microsomes.

Long-term stability of abused drugs and antiabuse chemotherapeutical agents stored at -20 degrees C.

Stability is an important consideration in the use of specimens for accurate determination of analyte concentrations. To determine the long-term stability for analytes routinely analyzed by mass spectrometry in this laboratory, quality-control (QC) results were plotted versus time. The time required for the initial concentration to reach a specified level of deviation (i.e., 15%) was then determined from the slopes. QCs were prepared at 1-3 concentrations in drug-free matrix and stored at approximately -20 degrees C; urines were fortified with 1% sodium fluoride; plasmas (except for cocaines) were prepared with heparin. For cocaine and metabolites, the plasma was either fortified with 2% sodium fluoride or with 2% sodium fluoride and 1 mg% physostigmine after adjustment of the plasma pH to 6.0. In urine, amphetamine, methamphetamine, codeine, morphine, benzoylecgonine (BZE), and 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCA) slopes did not exceed a 15% deviation before 852 days. Cocaine, however, reached a 15% reduction at 165 days. When cocaine and BZE were prepared in plasma with just 2% sodium fluoride, negative slopes reached 15% deviation within 154 and 111 days, respectively. Further fortification with physostigmide and adjustment of the pH extended this time frame significantly. Delta9-Tetrahydrocannabinol (THC) and THCA in plasma had negative slopes that deviated by 15% just prior to one year. l-Alpha-acetylmethadol (LAAM), methadone, and their N-demethylated metabolites in urine did not have any negative slopes exceeding 15% before 686 days. Several of the compounds had positive slopes. Those for 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine reached the 15% mark within 98 days. Those for LAAM, norLAAM, and dinorLAAM were concentration dependent. The 25-ng/mL controls reached 15% at 158-216 days. The 700-ng/mL controls reached 15% at 784-1340 days. In plasma, only naltrexone and buprenorphine displayed negative slopes at all three concentrations, reaching the 15% mark as early as 576 and 272 days, respectively. LAAM, norLAAM, dinorLAAM, ibogaine, 6-beta-naltrexone, risperidone, and 9-OH-risperidone did not exceed a 15% deviation before 416 days. To attempt to validate this method, two sets of clinical plasma samples that had been analyzed for buprenorphine were reanalyzed 644 and 869 days after the initial analyses. Those reanalyzed after 644 days were not statistically different from initial analyses, whereas those stored for 869 days were statistically different (p < 0.05). As the average time to reach 15% deviation for the three concentrations of buprenorphine QCs was 782 days, this suggests that extrapolation of QC results gathered over time may provide a reliable method to estimate long-term stability limits for drugs stored under the same conditions as the QC samples.

Determination of L-753,037 in human plasma by liquid chromatography/turbo ionspray tandem mass spectrometry.

A liquid chromatographic/turbo ionspray tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of L-753,037, a potent endothelin receptor antagonist currently under development for the treatment of cardiovascular diseases, in human plasma. L-753,037 is extracted from 0.5 ml of human plasma using liquid-liquid extraction and analyzed by LC/MS/MS with a turbo ionspray interface. Method validation results showed that this method is very sensitive, reliable, selective and reproducible. L-753,048, an ethoxy analogue of L-753,037, was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1). The intra-day precision and accuracy (n = 5) were measured to be below 10% relative standard deviation (RSD) and between 97.4 and 102.8% of the nominal values, respectively, for all calibration standard concentrations within the calibration curve range. The inter-day precision and accuracy (n = 3 days, 5 replicates per day) were measured to be below 6.5% RSD and between 99.3 and 102.0% of the nominal values, respectively, for all quality control concentrations. The extraction recovery was determined to be approximately 99% on average. The analyte was found to be stable in plasma through three freeze-thaw cycles, for at least 4 h at ambient temperature and for up to 40 days under -20 degrees C freezer storage conditions. The analyte was also shown to be stable for at least 24 h in the reconstitution solution at room temperature and for up to 3 days as a dried extract at 4 degrees C. Additional variations in plasma concentration of the analyte due to the use of different sources of plasma were also evaluated.

Sensitive triple-quadrupole mass spectrometric assay for the determination of BMS-181885, a 5-HT1 agonist, in human plasma following solid phase extraction.

A sensitive, selective, accurate, precise and reproducible triple-quadrupole liquid chromatographic-mass spectrometric assay was developed and validated for BMS-181885 (I), a 5HT1 agonist, in human plasma using BMS-181101 as the internal standard (IS). The method involved solid phase extraction of plasma containing I and the IS using Isoelute CN cartridges. The supernatant was then evaporated to dryness at 40 degrees C. The residue was dissolved in 100 microL of the injecting solvent. The HPLC column was ODS-3, 2 x 100 mm. The mobile phase comprised 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, used in an isocratic condition. The mass spectrometer was programmed to admit the protonated molecules at m/z 461 (I) and m/z 370 (IS) via the first quadrupole filter and to select reaction monitoring of ions at m/z 152 for I and IS for the quantification. Standard curves were fitted to a weighted quadratic function over the concentration range 0.2-200 ng/mL. The lowest standard concentration (0.2 ng/mL) was experimentally established as the lower limit of quantitation of the assay. The mean predicted quality control concentrations deviated within +/- 11% of the corresponding nominal values; the intra-assay and inter-assay precisions were within 7.0% relative standard deviation. I was stable in the injection solvent at 4 degrees C for at least 24 h and for at least three freeze-thaw cycles. Freezer stability of I in plasma was demonstrated for at least 3 months. The extraction recovery of I was established as 97%. The validated assay was applied to a pharmacokinetic study of I in humans.

Sensitive liquid chromatographic–mass spectrometric assay for the simultaneous quantitation of nefazodone and its metabolites hydroxynefazodone m-chlorophenylpiperazine and triazole-dione in human plasma using single-ion monitoring

A sensitive, selective, accurate, precise and reproducible high-performance liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the simultaneous determination of nefazodone (NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (I.S.). The method involved simultaneous protein precipitation with acetonitrile and liquid–liquid extraction with methylene chloride, after which the organic layer was evaporated to dryness. The residue was reconstituted in 25% acetonitrile in 10 m M ammonium formate (pH 4.0), and an aliquot was injected onto a BDS Hypersil C 18 column at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 m M ammonium formate (pH 4) and acetonitrile in 55:45 (v/v) was used in an isocratic condition. The mass spectrometer was programmed to admit the protonated molecules at m/ z 197.0 (mCPP), 372.0 (I.S.) 470.4 (NEF), 458.1 (Dione), and 486.2 (OH-NEF). Standard curves were linear ( r 2≥0.995) over the concentration range of 4–1000 ng/ml for Dione and 2–500 ng/ml for other analytes. The lowest standard concentrations were the lower limit of quantitation for each analyte. The mean predicted quality control (QC) concentrations for all analytes deviated less than −12.1% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay for all analytes were within 7.0% relative standard deviation. All analytes including I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 67.3 to 86.5%. The validated assay was applied to a pharmacokinetic study of nefazodone.

Quantitative subfemtomole analysis of alpha-tocopherol and deuterated isotopomers in plasma using tabletop GC/MS/MS.

A rapid, high-selectivity method with subfemtomole sensitivity is reported for quantification of alpha-tocopherol in plasma-based gas chromatography/tandem mass spectrometry (GC/MS/MS) using a tabletop quadrupole ion trap mass spectrometer. Sample workup is rapid, consisting of protein precipitation followed by liquid/liquid extraction and O-trimethylsilyl derivatization of alpha-tocopherol (alpha-T-TMS) and an internal standard, 2,2,5,7,8-pentamethyl-6-chromanol (PC-TMS). Rudimentary chromatography was carried out using an 8-m DB-5 capillary column resulting in an analyte retention time of 7.2 min. No interferences from the plasma matrix were observed. The assay has a detection limit of 178 amol (89.6 fg) and a lower limit of quantification of 700 amol (350 fg) of derivatized alpha-tocopherol in diluted plasma; < 30 pL of plasma is estimated to yield sufficient alpha-tocopherol for quantitative analysis at typical concentrations found in humans. A calibration curve constructed from National Institute of Standards and Technology serum standards was linear in the working range of 1.9-1073 ng/mL (0.95-0.54 ng). Within- and between-day precision averaged 5.8% and did not exceed 11.3% for three concentrations of quality control (QC) solutions. The overall accuracy for the QC samples was within 7.2%. Storage studies showed that, alpha-T-TMS and PC-TMS are stable under conditions that might be encountered during analyses. In a test study, plasma kinetic curves for alpha-tocopherol-d6 and alpha-tocopherol-d3 were obtained for a catheterized pregnant ewe and her fetus who were simultaneously given a bolus injection of alpha-tocopherol-d6, to the ewe and alpha-tocopherol-d3 to the fetus. These data show that a tabletop GC ion trap can determine alpha-T-TMS and its isotopomers quantitatively at high selectivity in a complex matrix.

Improved Solid-Phase Extraction Technique for Plasma Flecainide Analysis by High-Performance Liquid Chromatography

A method for analyzing flecainide in plasma was developed and assessed. Based on the use of C18 extraction columns eight or fewer times, the rapid and simple extraction procedure provided consistent, high-efficiency flecainide extraction (>85%). Using reverse-phase, high-performance liquid chromatography with fluorometric detection, flecainide acetate was detectable to approximately 15 ng/ml. Retention times of the internal standard and flecainide were 8.9 to 9.2 and 9.8 to 10.4 minutes, respectively, and short sample preparation and run times enabled results to be delivered within 2 to 3 hours of receiving samples. The assay was linear for the standard range 20 ng/ml to 2 microg/ml (r2 with three standards, >0.999) and delivered a high level of accuracy. Quality control concentrations obtained from eight assays consistently fell within 5% of nominal values (100 ng/ml and 1 microg/ml). Based on four assays, the assay was also reproducible with calculated-between and within-assay coefficients of variation of less than 1% and 3%, respectively. The authors found that the performance of this assay was excellent and that the solid-phase extraction technique was simple, rapid, and cost effective.

Determination of the dihydrocodeine metabolites, dihydromorphine and nordihydrocodeine, in hepatic microsomal incubations by high-performance liquid chromatography

A high-performance liquid chromatography assay for the oxidative metabolites of dihydrocodeine, nordihydrocodeine and dihydromorphine, formed in human liver microsomal incubations, is described. A simple solvent extraction followed by reversed-phase high-performance liquid chromatography with UV detection allows quantification of both metabolites in a single assay. Standard curve concentration ranges for dihydromorphine and nordihydrocodeine were 0.05–5 and 0.2–20 μM, respectively. Assay performance was assessed by intra- and inter-day accuracy and precision of quality control (QC) samples. The difference between the calculated and the actual concentration and the relative standard deviation were less than 15% at low QC concentrations and less than 10% at medium and high QC concentrations for both analytes. The method provides good precision, accuracy and sensitivity for use in kinetic studies of the oxidative metabolism of dihydrocodeine in human liver microsomes.