Rapid method for the simultaneous measurement of nicotine and cotinine in urine and serum by gas chromatography–mass spectrometry

A simple, sensitive, and rapid gas chromatographic–mass spectrometric method is described for the simultaneous detection and quantitation of nicotine and its metabolite, cotinine, in urine and serum. The analytes and their respective deuterated internal standards were extracted by liquid–liquid extraction coupled to centrifugation and evaporation. The detection limit of the assay was 0.16 ng/ml for both nicotine and cotinine. The limit of quantitation for each analyte was 1.25 ng/ml.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Determination of four carboxylic acid metabolites of felodipine in plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid—liquid and solid-phase extraction. The metabolites were applied to a C₂ cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C₁₈ column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration—time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.     

Methods for the determination of seven selective serotonin reuptake inhibitors and three active metabolites in human serum using high-performance liquid chromatography and gas chromatography

This paper describes a set of simple and sensitive multiresidue methods for the determination of the specific serotonin reuptake inhibitors (SSRIs) used as antidepressant drugs, and some of their respective active metabolites in human serum. It involves liquid–liquid extraction procedures followed by gas chromatography coupled to nitrogen phosphorus detection or isocratic reversed-phase high-performance liquid chromatography combined with fluorescence detection (HPLC–FL), depending on the analytes. Extraction recoveries were between 71 and 96% for the eight SSRIs and their metabolites analysed by GC and between 41 and 77% for the two of them analysed by HPLC. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 2.5 to 5 μg/l and from 10 to 20 μg/l. Intra-assay and inter-assay precision was studied at three and four concentration levels, respectively, and was less than 19% for all compounds. Accuracy was also satisfactory for all. An excellent linearity was observed from the LOQs up to 1000 μg/l for milnacipram and paroxetine and from each LOQ up to 400 mg/l for the other compounds. The performance of the methods described thus allows the therapeutic drug monitoring of the currently commercialised SSRIs.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique

Fluorimetric column-switching HPLC method with naphthalene-2,3-dialdehyde (NDA) was developed for the determination of endogenous angiotensin (ANG) metabolites in human plasma. After one-step extraction to clean up the ultrafiltered plasma sample on the reversed HPLC system, the zone of the retention time of each ANG analyte was subjected to the NDA-derivatization. After putting into a first Phe-ODS (for ANG (3–4) and (5–8) determinations) or ODS column (for ANG I and II determinations), the heart-cut of the retention time of the NDA-ANG was separated on a second ODS column with a mobile phase containing 5 mM ion-pair reagent. Complete separation and good detection were accomplished within 2 h. Good linearity of the regression equation for all ANG analytes with the correlation coefficient of >0.993 as well as good reproducibility (C.V.<4.0%). Good agreement of the range of ANG II plasma level between the present (25–47 fmol/ml in plasma) and the radioimmunoassay methods (28–52 fmol/ml in plasma) indicated that the column-switching method could be applicable for the determination of endogenous smaller ANGs as well as for ANG I or II in plasma.     

Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride

An improved HPLC method using a silica gel column with fluorescence detection (excitation at 300 nm and emission at 365 nm) was developed for the determination of sulpiride concentrations in plasma. Analysis of sulpiride in plasma samples was simplified by a one-step liquid–liquid extraction after alkaline treatment of only 1 ml of plasma. The low limit of quantitation was 20 ng/ml with a coefficient of variation of less than 20%. A linear range was found from 20 to 1500 ng/ml. This HPLC method was validated with the precision for inter-day and intra-day runs being 0.36–8.01% and 0.29–5.25%, respectively, and the accuracy (standard deviation of mean, SD) for inter-day and intra-day runs being −1.58 to 5.02% and −2.14 to 5.21%, respectively. Bioequivalence of the two products was evaluated in 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment cross-over study. Sulpiride plasma concentrations were analyzed with this validated HPLC method. Results demonstrated that the two tablet formulations of sulpiride appear to be bioequivalent.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

Validated method for the therapeutic drug monitoring of flunitrazepam in human serum using liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry with an ion trap detector

An HPLC–MS–MS method for the quantitative analysis of flunitrazepam in human serum was established. The method features a very simple liquid–liquid extraction, the use of a standard 4-mm HPLC column, isocratic elution using a buffer-free solvent, short retention times in connection with good peak resolution and the sensitivity and selectivity of an ion trap MS–MS detector. The procedure enables unambiguous identification of analytes by their product ion spectra, as well as sensitive quantitation (limit of quantitation for flunitrazepam=0.5 ng/ml). This feature was used for the confirmation of HPLC–UV results for nitrazepam.     

High-performance liquid chromatographic method for the simultaneous determination of nalbuphine and its prodrug, sebacoyl dinalbuphine ester, in dog plasma and application to pharmacokinetic studies in dogs

For the determination of nalbuphine and its long acting prodrug, sebacoyl dinalbuphine ester (SDN), in biological samples, a reversed-phase high-performance liquid chromatographic method using dual detectors was established. Ultraviolet and fluorescence detectors were connected in series for determining SDN and nalbuphine, respectively. The two analytes and internal standard were extracted from plasma by alkaline liquid–liquid extraction using n-hexane–isoamyl alcohol (9:1, v/v). The calibration curve for nalbuphine was linear over the range from 10 to 2500 ng/ml, while the range was 25 to 2500 ng/ml for SDN. The within- and between-day precision and accuracy were all within 10% for both nalbuphine and SDN over these concentrations. The method was applied successfully to a pharmacokinetic study of SDN administered at 20 mg/kg to two beagle dogs. Pharmacokinetic analysis revealed that SDN followed a linear one-compartment model with an elimination half-life of 74.7 min. Formation of nalbuphine after intravenous administration of SDN was observed in the first time point sample (5 min). These results indicate that SDN is rapidly metabolized to its active moiety, nalbuphine, in dogs and no other metabolites are detected in plasma.     

Antiatherogenic effects of cilostazol and probucol alone, and in combination in low density lipoprotein receptor-deficient mice fed with a high fat diet.

Cilostazol, an antiplatelet drug, and probucol, a cholesterol-lowering drug, are reported to ameliorate atherosclerosis in animal models. However, their combined effect on atherosclerosis is unclear. We therefore evaluated their combined effect on atherosclerotic lesions in LDL receptor-deficient mice. Male LDL receptor-deficient mice were fed a high fat diet with or without cilostazol alone, probucol alone, or with cilostazol and probucol in combination, for 8 weeks. Body weight and plasma lipid levels were measured before and during treatment. At the end of treatment, the size distribution of plasma lipoproteins was analyzed by HPLC and then plasma HDL cholesterol levels and en face aortic atherosclerotic lesion areas were measured. Probucol alone significantly decreased both total cholesterol and HDL cholesterol, while cilostazol alone did not decrease total cholesterol, but significantly increased HDL cholesterol. Both cilostazol alone and probucol alone significantly decreased atherosclerotic lesion areas, and their combined administration showed more significant decreases than when each drug was administered singly. The combination of cilostazol and probucol was more effective in preventing atherosclerotic lesion formation than the administration of each drug alone; this may provide us with a new strategy for treating atherosclerosis.     

Determination of amide-type local anaesthetics by direct injection of plasma in a column-switching high-performance liquid chromatographic system using a pre-column with a semipermeable surface

A high-performance liquid chromatographic method using column switching was applied to the direct determination of two local anaesthetics, ropivacaine and bupivacaine, in human plasma. The method is intended to be used in a combined LC—GC system; here only the LC-part is described. After addition of internal standard, the samples were filtered and directly injected into a semipermeable surface (SPS) pre-column where the analytes were strongly retained and separated from many endogenous compounds by a short washing step. The retained analytes were transferred by a buffered methanol phase from the pre-column into a carbonaceous HPLC column and they were detected by UV detection at 254 nm. The SPS pre-column could withstand numerous (> 200) direct injections of plasma samples (10 μl). The method has a detection limit of 8.2 ng and requires a total assay time of 15 min per plasma sample. Quantitative recoveries were obtained over the range 3.3–114 μg/ml with inter-day precisions of 1.6–5.2% (C.V.).     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

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.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid—liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3–0.8 pg injected) and reproducible (coefficients of variation 1–10%), and shows good accuracy (93–98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

Determination of moxifloxacin (BAY 12-8039) in plasma and lung tissue by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method with a new polymeric cartridge

The aim of this study was to develop a high-performance liquid chromatographic (HPLC) assay for the determination of moxifloxacin in human plasma and lung tissue. The assay was based on HPLC with a Supelcosil ABZ+ column and ultraviolet detection set at a wavelength of 296 nm. The extraction procedure was characterized by a fully automated liquid–solid extraction using an OASIS column for the solid phase. The assay has been found to be linear and validated over the concentration range 3.2 to 0.025 μg/ml for moxifloxacin in plasma and from 16 to 0.25 μg/g for moxifloxacin in lung tissue. In future, the assay will support the pharmacokinetic study of the penetration of moxifloxacin in human lung tissue.     

Cilostazol Attenuates Ovariectomy-Induced Bone Loss by Inhibiting Osteoclastogenesis

BackgroundCilostazol has been reported to alleviate the metabolic syndrome induced by increased intracellular adenosine 3’,5’-cyclic monophosphate (cAMP) levels, which is also associated with osteoclast (OC) differentiation. We hypothesized that bone loss might be attenuated via an action on OC by cilostazol.Conclusions/SignificanceOur data highlight the therapeutic potential of cilostazol for attenuating bone loss and oxidative stress caused by loss of ovarian function.