High-performance liquid chromatography–electrospray ionization mass spectrometry method for the measurement of moclobemide and two metabolites in plasma

A rapid and sensitive liquid chromatography–electrospray ionisation mass spectrometry (HPLC–ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C₁₈ cartridges. A Nova-Pak phenyl column (Waters, 4 μm, 150×2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 μg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.     

Multi-analyte analysis of biological fluids with a recycling immunoaffinity column array.

A system for isolating and measuring up to 30 analytes in a single biological sample is described. The system is based on recycling a pre-labeled sample through an array of capillary immunoaffinity columns, each packed with glass beads, coated with a different antibody, thus enabling each column to isolate and extract a single analyte. Detection of the bound analytes is achieved by laser-induced fluorescence (LIF), using a laboratory-built scanning detector coupled to a fiber-optic spectrometer. The array can be regenerated up to 200 times, provided a suitable temperature is maintained. The individual immunoaffinity columns are able to bind between 2.9 and 3.6 ng of analyte, depending upon the individual column, with lower limits of detection (LOD) in the order of 1.6-2.8 pg/ml. The inter- and intra-assay coefficients of variation (CV) for all 30 columns in the array were less than 6.03+/-0.33 at analyte concentrations of 100 pg/ml. Comparison to standard enzyme-immunoassays demonstrated r(2) values in the range of 0.9151-0.9855 when analyzed by least-squares linear regression.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.     

High-performance liquid chromatographic determination of 1-β- -arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum

A high-performance liquid chromatographic method was developed to assay 1-β- -arabinofuranosyl-E-5-(2-bromovinyl)uracil and its metabolite (E)-5-(2-bromovinyl)uracil in serum. The chloro analogue of the parent drug is used as internal standard. Human serum samples were assayed to establish the pharmacokinetic parameters. Acetonitrile, used as a protein precipitant, was evaporated to dryness and the residue, containing the analytes and internal reference, was dissolved in mobile phase prior to chromatographic analysis. The minimum quantifiable level was 0.02 μg of each analyte per ml of serum.     

Accelerated solvent extraction and liquid chromatography–tandem mass spectrometry quantitation of corticosteroid residues in bovine liver

A new method for the rapid extraction and unequivocal confirmation of two highly potent fluorinated synthetic corticosteroids, dexamethasone and its β-epimer betamethasone, in bovine liver was developed. Flumethasone was used as internal standard. An extraction procedure using an accelerated solvent extraction system was employed for the isolation of the analytes in liver samples. The procedure was highly automated, including defatting and extraction steps, sequentially carried out under 1.0·10⁴ kPa in about 35 min. The extracts were then directly analysed by tandem mass spectrometry with on-line liquid chromatography. The analytes were ionised in a heated nebulizer interface operating in the negative ion mode where the molecular related ions [M-H-CH₂O]⁻ were generated for each analyte, at m/z 361 for betamethasone and dexamethasone and at m/z 379 for flumethasone. They served as precursor ions for collision-induced dissociation and three diagnostic product ions for the drugs were identified to carry out analyte confirmation by selected reaction monitoring. Assessment of recovery, specificity and precision for betamethasone, dexamethasone and flumethasone proved the method suitable for confirmatory purposes. The limit of quantification of betamethasone and dexamethasone in liver tissue was 1.0 μg/kg.     

Determination of risperidone and enantiomers of 9-hydroxyrisperidone in plasma by LC-MS/MS

A robust and validated liquid-liquid extraction LC-MS/MS method was developed for population pharmacokinetic analysis and therapeutic drug monitoring of risperidone and the enantiomers of its major active metabolite (+)-and (-)9-hydroxyrisperidone in pediatric patients. The method was rapid, sensitive and used a low sample amount (200 microL), which is very desirable for the pediatric population. The assay was validated from 0.2 to 50 ng/mL in plasma for all analytes. LLOQ for all analytes was 0.2 ng/mL. The extracts were analyzed by normal phase LC-MS/MS. The sample run time was 8 min. Intra- and interday precision for all analytes was < or =6%; method accuracy was between 89 and 99%. Additional experiments were performed to analyze matrix effects and identify a proper internal standard for each analyte. The validated method was used to study risperidone and its enantiomer metabolites in plasma as part of a population pharmacokinetic study in pediatric patients with pervasive developmental disorder (PDD).     

Simultaneous measurement of the cell-differentiating agent hexamethylene bisacetamide and its metabolites by gas chromatography

Hexamethylene bisacetamide (HMBA) is a potent in vitro differentiating agent that has clinical potential as an anticancer drug both as a single agent and as a component of combination therapy. A sensitive and efficient GC method for the isolation, derivatization, and measurement of both HMBA and its two major metabolites in plasma and urine in a single analysis is described. In situ carbamylation of the biological sample with diethylpyrocarbonate forms the urethane derivative of the basic N-acetyl diaminohexane metabolite and allows analyte isolation and concentration by solid-phase extraction. Subsequent formation of the n-butyl ester of 6-acetamidohexanoic acid, the major metabolite, provides a derivatized biological extract that can be rapidly analyzed by temperature-programmed GC. The quantitative extraction and the efficient derivatization steps provide a limit of quantitation of 0.05 mM (10 μg/ml) for all analytes with a precision better than 8% for the range of in vitro activity (0.1–2.0 mM). This method is amenable to automation and is well-suited for the analysis of clinical samples.     

Microwave-assisted acid extraction methodology for trace elements determination in mastic gum of Pistacia lentiscus using inductively coupled plasma atomic emission spectrometry

Introduction – To ensure food safety, accurate knowledge of the levels of several trace elements is necessary. This is also true for natural products of plants and resins used for human consumption or therapeutic treatment, like the mastic gum of Pistacia lentiscus. The rapid analysis of gum and resin matrices is a challenge because there are problems with the decomposition of such complicated matrices. Objective – To develop an efficient multielemental analytical method for the determination of trace elements and to compare different procedures for analyte extraction when microwave‐assisted digestion is applied. Methodology – The inductively coupled plasma atomic emission spectrometric (ICP‐AES) technique was applied and the optimum ICP conditions like radiofrequency power, argon flow rate and nebuliser sample uptake flowrate were found. The microwave‐assisted procedure was compared with that with conventional heating. Since mastic and resinous materials are difficult for dissolution and extraction of trace element, influential acid mixtures containing hydrofluoric acid proved to be capable of quantitative extraction of the analytes. Results – The digestion of mastic resin or similar matrices is significantly facilitated by using microwave radiation instead of conventional heating since the obtained recovery for several analytes is much higher. It was proved that the acid mixture of HCl–HNO₃–HF was the most efficient for complete sample digestion and recovery of the analytes. Conclusions The performance characteristics of the developed method were evaluated against certified reference material and the method was proved reliable and applicable to the analysis of mastic gum and possibly to similar resinous matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatographic–electrospray tandem mass spectrometric method for the simultaneous quantitation of the prodrug fosinopril and the active drug fosinoprilat in human serum

A sensitive, specific, accurate and reproducible LC–MS–MS method was developed and validated for the simultaneous quantitation of the prodrug fosinopril and its active drug fosinoprilat in human serum. The method employed acidification of the serum samples to minimize the hydrolysis of fosinopril to fosinoprilat prior to purification by solid-phase extraction to isolate the two analytes and the two internal standards from human serum. The extracted samples were analyzed by turbo ionspray LC–MS–MS in the positive ion mode. Chromatography was performed on a polymer-based C₁₈ column (Asahipak™ ODP PVA-C₁₈, 2×50 mm) using gradient elution with methanol and 10 mM ammonium acetate, pH 5.5. The calibration curve, 1.17 to 300 ng/ml, was fitted to a weighted (l/x) linear regression model. Serum quality control (QC) samples used to gauge the accuracy and precision of the method were prepared at concentrations of 5.00, 100, 250 and 500 ng/ml of each analyte. The inter-assay accuracies were within 6% (DEV) for both analytes. The intra- and inter-assay precisions were within 7% and 11% (RSD), respectively, for both analytes. The hydrolysis of fosinopril to fosinoprilat during sample processing was ≤6%. This degree of conversion would cause little error in the analysis of post-dose serum samples since such samples are known to contain low levels of the prodrug compared to the drug.     

Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 μl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 μm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 μg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose™ formulation.     

Bioanalysis of aplidine, a new marine antitumoral depsipeptide, in plasma by high-performance liquid chromatography after derivatization with trans-4′-hydrazino-2-stilbazole

A sensitive bio-analytical assay in plasma of the depsipeptide aplidine is reported, based on reversed-phase liquid chromatography and fluorescence detection of the trans-4′-hydrazino-2-stilbazole (4′H2S) derivative of the analyte. At ambient temperature, two conformations of the depsipeptide are observed in solution due to cis–trans isomerism at the proline–pyruvoyl peptide bond. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, a derivatization with 4′H2S is performed in a water–acetonitrile mixture at pH 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2–100 ng/ml-range, 2 ng/ml being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The identity of the 4′H2S reaction products of aplidine have been confirmed by mass spectrometric analysis. Finally, the method has been employed for a pilot pharmacokinetic study of aplidine in mice which demonstrated its usefulness for pharmacological research.     

Arginine and methylated arginines in human plasma and urine measured by tandem mass spectrometry without the need for chromatography or sample derivatisation

A method for the simultaneous analysis of asymmetric dimethylarginine, symmetric dimethylarginine, monomethylarginine and arginine in human plasma and urine, with short analysis time and isotopic internal standardisation for each analyte is described. The method requires neither sample derivatisation nor the need for chromatographic separation of analytes. The method described shows good precision and accuracy and is suited for both research purposes and implementation in the busy, routine clinical laboratory. In addition the synthesis and utilisation of isotopically labelled symmetric dimethylarginine and monomethylarginine is described for the first time, avoiding the use of surrogates such as homoarginine for internal standardisation.     

A chiral stationary phase which affords unusually high levels of enantioselectivity

A chiral stationary phase (CSP) derived from N-(1-naphthyl) leucine has been prepared. This CSP is conceptually similar to the CSP derived from N-(2-naphthyl)alanine and was expected to separate the enantiomers of the same clientele of analytes as does the latter. The magnitudes of the separation factors observed on the two CSPs may differ markedly for a given analyte, the new CSP often affording much greater enantioselectivity.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Stereoselective measurement of E- and Z-doxepin and its N-desmethyl and hydroxylated metabolites by gas chromatography–mass spectrometry

A stereoselective method of analysis of the antidepressant drug doxepin (DOX, an 85:15% mixture of E–Z stereoisomers), its principal metabolites E- and Z-N-desmethyldoxepin (desDOX) and ring-hydroxylated metabolites in microsomal incubation mixtures is described. DOX and its metabolites were extracted from alkalinised incubation mixtures by either: 9:1 hexane–propan-2-ol (method 1) or 1:1 hexane–dichloromethane (method 2), derivatised with trifluoroacetic anhydride and analysed by GC–MS with selected ion monitoring. Both methods were suitable for the analysis of individual desDOX isomers as indicated by correlation coefficients of ≥0.999 for calibration curves constructed between 50 and 2500 nM, and good within-day precision at 125 nM (C.V. ≤14%) and 1000 nM (C.V. ≤8%). Method 1, however, was unsuitable for the analysis of ring-hydroxylated metabolites of DOX, whereas the hydroxylated metabolites of E-DOX and E-desDOX (generated in situ) were extracted by method 2 with a C.V. of ca. 13%. This is the first assay method that permits the simultaneous measurement of desDOX and hydroxylated metabolites of DOX in microsomal mixtures.     

Band centrifugation of macromolecules in self-generating density gradients. II. Sedimentation and diffusion of macromolecules in bands

Three approaches to the simultaneous sedimentation and diffusion of hands or zones of noninteracting homogeneous macromolecules are examined: (1) The authors' method of moments: (2) the transport me of Sehumaker and Rosenbloom; and (3) the stochastic solution of the Lamm equation due to Gehatia and Katehalski. All three methods indicate that the motion of the maximum of the hand may be used to evaluate the sedimentation coefficient. The moment, method provides relations which appear to be useful for measuring diffusion coefficients. Relations are given for the analysis of resolved components. The problem of measuring sedimentation coefficients of macromolecules with concentration-dependent sedimentation coefficients is examined. Methods are described for evaluating the sedimentation coefficient in these systems and for obtaining the sedimentation coefficient at infinite dilution. Methods are described for determining the weight-average sedimentation coefficient in Multi-component systems, and the differential and integral distribution of sedimentation coefficients of macromolecules with low-diffusion coefficients.     

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.     

A gas chromatography-mass spectrometry method for the measurement of fatty acid omega and omega(-1) hydroxylation kinetics by CYP4A1 using an artificial membrane system

A gas chromatography-mass spectrometry assay method for the analysis of lauric, myristic, and palmitic acids and their omega and omega(-1) hydroxylated metabolites from in vitro incubations of cytochrome P450 CYP4A1, involving solid-phase extraction and trimethysilyl derivatization, was developed. The assay was linear, precise, and accurate over the range 0.5 to 50microM for all the analytes. It has the advantages of a more rapid analysis time, an improved sensitivity, and a wider range of analytes compared with other methods. An artificial membrane system was optimized for application to purified CYP4A1 enzyme by investigating the molar ratios of cytochrome b(5) and cytochrome P450 reductase present in the incubation mixture. Using this method, the kinetics of omega and omega(-1) oxidation of lauric, myristic, and palmitic acids by CYP4A enzymes were measured and compared in rat liver microsomes and an artificial membrane system.     

Determination of epimers 22R and 22S of budesonide in human plasma by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A highly sensitive and selective method has been developed for the simultaneous quantification of 22R- and 22S-epimers of budesonide in human plasma. The drug was isolated from human plasma using C₁₈ solid-phase extraction cartridges and was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives. Deuterium-labelled budesonide was synthesized and determined to have an isotopic purity > 99%. This was used as the internal standard. Epimers were quantified by automated liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, operating in selected ion mode at m/z 473.2 and m/z 476.2. Linear responses were observed for both epimers over the range 0.25 to 10.0 ng/ml. The average recoveries of 22R- and 22S-epimers of budesonide from human plasma were 87.4% and 87.0%, respectively. The lower limit of quantification for each epimer was 0.25 ng/ml, corresponding to 50.0 pg of analyte on column. Within- and between-day coefficients of variation were 8.6% and 4.0%, respectively.