High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.     

Simultaneous determination of lansoprazole enantiomers and their metabolites in plasma by liquid chromatography with solid-phase extraction

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5M NaClO(4)-acetonitrile-methanol (6:3:1 (v/v/v)). The analysis required only 100 microl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10-1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.     

Rapid high-performance liquid chromatographic measurement of amisulpride in human plasma: application to manage acute intoxication

Amisulpride, a substituted benzamide derivative, is a second-generation (atypical) antipsychotic and is effective as maintenance therapy in patients with schizophrenia. For toxicological purpose, a rapid RP-HPLC assay was developed for the determination of amisulpride in human plasma. A linear response was observed over the concentration range 100-1000 ng/ml. A good accuracy (< or =5%) was achieved for all quality controls, with intra- and inter-day variation coefficients equal or inferior to 4.9%. The lower limit of quantification was 20 ng/ml, without interferences of endogenous components. This rapid method (run time <5 min) was used to monitor eight intoxications involving amisulpride.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma

A reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra? MS C(18) column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5-2000 ng/ml in plasma for the total drug and 0.5-200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.     

Determination of CGS 16617 and stable isotope-labeled CGS 16617, an angiotensin-converting enzyme inhibitor, in human plasma by gas chromatography/mass spectrometry

An analytical method has been developed for the simultaneous determination of a novel orally active angiotensin-converting enzyme inhibitor (CGS 16617) and a stable isotope-labeled analog. Both compounds are isolated from human plasma using an ion-exchange column, derivatized with pentafluoropropionic anhydride and pentafluoropropanol, and analyzed by gas chromatography/mass spectrometry. After splitless injection on a methyl-silicon column, the compound is detected using negative ion chemical ionization with nitrous oxide as a reagent gas. CGS 16617 labeled with four deuteriums and two 13C is used as an internal standard. The accuracy and precision of the method, expressed as the overall mean +/- SD recovery obtained from two sets of 36 quality-control samples used during a clinical study (concentration range 0.2-100 ng ml-1 plasma), was 96.1 +/- 16.2% for unlabeled drug and 97.6 +/- 14.4% for the D4-labeled drug (concentration range 0.2-100 ng ml-1 plasma). The limit of quantification using 1 ml plasma is 0.2 ng ml-1 for both labeled and unlabeled drug.     

Determination of a potential anxiolytic drug (CGS 20625) in human plasma by high-performance liquid chromatography

An analytical method employing reversed-phase high-performance liquid chromatography is described for the determination of a potential anxiolytic agent in human plasma. This experimental drug candidate has potent and selective affinity for the central benzodiazepine receptor complex. The compound and internal standard are extracted from buffered plasma (pH 9.0) into ethyl acetate. The solvent is evaporated and the residue is reconstituted in chromatographic mobile phase. Separation is achieved on a 5-μm phenyl column with ultraviolet absorbance detection of the drug and internal standard at 270 nm. Recovery and reproducibility assessments indicate good accuracy (overall relative recovery of 101%) and precision (coefficients of variation from 2.0 to 11%) over the concentration range 10–1000 ng/ml. The limit of quantification for the method is 10 ng/ml. The method is suitable for pharmacokinetic analysis following the administration of 80 mg of drug to normal volunteers.     

Sensitive method for the assay of sertindole in plasma by high-performance liquid chromatography and fluorimetric detection

A simple and highly sensitive normal-phase HPLC method is described for determining sertindole concentrations in human plasma using fluorimetric detection. A short C₈ column was used to extract sertindole and the internal standard from plasma; the column was rinsed with acetonitrile, and the analytes were recovered by elution with methanol. This uncommon selectivity between the two solvents allowed clean extraction and near- quantitative recovery of the analytes (> 89%). Separation was done on a 5-μm silica-gel column and detection was performed by fluorimetry, with emission at 340 nm and excitation at 260 nm. The detection and lower quantifiable limits were 0.01 and 0.025 ng/ml, respectively, with no interference from plasma or potential metabolites.     

Simultaneous determination of haloperidol and bromperidol and their reduced metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography

This study describes a new simultaneous determination of haloperidol and bromperidol and their reduced metabolites by modification of automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1ml of plasma using chloroform-hexane (30:70 (v/v)), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C(18) analytical column for separation. The mobile phases consisted of phosphate buffer (0.02M, pH 4.6), perchloric acid (60%) and acetonitrile (54:1:45 (v/v)) and was delivered at a flow-rate of 0.6ml/min. The peak was detected using a UV detector set at 215nm. The method was validated for the concentration range 1-100ng/ml, and good linearity (r >0.999) was confirmed. Intra-day coefficient variations (CVs) for haloperidol, reduced haloperidol, bromperidol and reduced bromperidol were less than 2.5, 3.1, 2.4 and 2.5%, respectively. Inter-day CVs for corresponding compounds were 3.9, 5.1, 2.6 and 4.4%, respectively. Relative errors ranged from -5 to 10% and mean recoveries were 96-100%. The limit of quantification was 1.0ng/m for each compound. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring, particularly in patients receiving both haloperidol and bromperidol.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction

A new method is described for the determination of cimetidine in human plasma. The drug and internal standard (ranitidine) were separated on a Nucleosil C₁₈ 5 μm (25 × 4.6 mm I.D.) column using a mobile phase of acetonitrile-phosphate buffer, pH 6.2 (25:75, v/v) containing 2.5 g/l heptane sulphonic acid. The mobile phase was delivered at a flow-rate of 0.9 ml/min, detection was by ultraviolet absorption at 228 nm and concentrations were calculated on the basis of peak areas. The drugs were extracted from alkaline plasma into ethyl acetate using a salting out procedure which involved the addition of 100 ml of a saturated solution of K₂CO₃ to each 250-μl plasma aliquot. The method was validated over the concentration ranges 50–3000 ng/ml and 100–7000 ng/ml for two separate studies. Mean coefficients of variation were less than 6% for both intra- and inter-assay in both studies and recoveries varied between 71 and 81%. The method was successfully applied to the determination of cimetidine in plasma for a pharmacokinetic study.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Determination of unbound ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS/MS

Ticagrelor is the first direct acting reversibly binding oral platelet P2Y(12) receptor antagonist. The parent molecule and the main metabolite (AR-C124910XX) are both able to block adenosine diphosphate-induced receptor signaling with similar potency. Drug binding to plasma proteins reduces free drug available for pharmacologic activity. Therefore, assessing unbound drug is important for interpretation of pharmacokinetic/pharmacodynamic findings. This paper describes the development and validation of an equilibrium dialysis/LC-MS/MS method for measuring unbound ticagrelor and AR-C124910XX in human plasma. Plasma samples (200μl) were dialysed against phosphate buffered saline (37 °C, 24h) in 96-well dialysis plates to separate unbound analytes. Drug-protein binding alterations during dialysis were minimized by maintaining physiologic conditions (pH 7.4, 37 °C). Ticagrelor and AR-C124910XX were quantified in dialysates (unbound fraction), retentates and plasma (total concentration) using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods. Calibration curves were established for the retentate and plasma (total concentration) in the ranges 5-5000 ng/ml (ticagrelor) and 2.5-2500 ng/ml (AR-C124910XX), and for the dialysate in the range 0.25-100 ng/ml (both analytes). Both ticagrelor and AR-C124910XX were highly protein bound (>99.8%), i.e. unbound fraction <0.2%. Yet, the methodology was successfully applied to determine unbound concentrations of ticagrelor and AR-C124910XX in clinical samples.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Uptake and effect of praziquantel and the major human oxidative metabolite, 4-hydroxypraziquantel, by Schistosoma japonicum

After exposure to praziquantel in vitro at a concentration of 1 microgram/ml for 0.5-2 hr, amounts of praziquantel in Schistosoma japonicum varied from 2.1 +/- 1.2 to 3.7 +/- 1.6 ng/male worm and 1.3 +/- 1.2 to 2.2 +/- 1.5 ng/female worm during the time studied. At 30 micrograms/ml, praziquantel amounts were 11-33-fold higher. However, within 2 hr after removal from a medium containing 30 micrograms/ml praziquantel, 95% of the drug was released from the parasites. When S. japonicum worm pairs were incubated in vitro with 1, 10, and 30 micrograms/ml of 4-hydroxypraziquantel, the major human oxidative metabolite of praziquantel, 0.2 +/- 0.2, 3.8 +/- 1.3, and 7.4 +/- 1.3 ng/worm pair, respectively, were found after a 2-hr incubation. 15-30-fold lower than corresponding worm pair amounts of praziquantel. In vivo, when 4- or 5-wk S. japonicum-infected mice were treated orally with praziquantel (300 mg/kg), peak concentrations of praziquantel in plasma determined by high pressure liquid chromatography were 14.7 +/- 1.5 micrograms/ml (4-wk infection) and 16.7 +/- 2.8 micrograms/ml (5-wk infection) 15 min after treatment. Corresponding in vivo worm praziquantel amounts were 1.8 +/- 0.4 ng/male worm and 2.4 +/- 1.1 ng/female worm, respectively, in the 4-wk infection and 4.6 +/- 1.6 ng/male worm and 5.6 +/- 1.2 ng/female worm in the 5-wk infection. Peak plasma concentrations of 4-hydroxypraziquantel were similar but corresponding in vivo worm amounts were 1-20-fold lower, depending on the time after drug administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Determination of a new atypical antipsychotic agent perospirone and its metabolite in human plasma by automated column-switching high-performance liquid chromatography

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the quantification of perospirone, a serotonin and dopamine antagonist, and its metabolite ID-15036 in human plasma. The test compounds were extracted from 2 ml of plasma using chloroform-hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 micro m, 35 x 4.6 mm I.D.) for clean-up and column II (C(18) STR ODS-II analytical column, 5 micro m, 150 x 4.6 mm I.D.) for separation. The peak was detected using a fluorescence detector set at Ex 315 nm and Em 405 nm, and the total time for a chromatographic separation was approximately 30 min. The method was validated for the concentration range from 0.1 to 100 ng/ml. Mean recoveries were 97% for perospirone and 96% for ID-15036. Intra- and inter-day relative standard deviations were less than 2.8 and 5.3% for perospirone and 2.4 and 4.4% for ID-15036, respectively, at the concentration range from 0.3 to 30 ng/ml. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in human plasma. An Oasis((R)) HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 microm, 120A, [Formula: see text] mm) column. The initial composition of the mobile phase was 2% methanol/98% 5mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3min, followed by a linear gradient over 4 min to 30% methanol/70% 5mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5'-deoxy-5-fluorouridine (5'-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC-H](-), m/z 263 for [dFdU-H](-), and m/z 245 for [5'-DFUR-H](-). The average recoveries for dFdC, dFdU, and 5'-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5-1000 ng/ml for dFdC, and 5-5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were 相似文献