Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.     

Rapid and simple micro-determination of carvedilol in rat plasma by high-performance liquid chromatography

We studied the use of high-performance liquid chromatography (HPLC) with spectrofluorometric detection, using a solid-phase extraction for a simple, rapid and sensitive determination of plasma carvedilol levels in rats. Extracted aliquots were analyzed by HPLC, using a reversed-phase octadecyl silica column. The analytical mean recovery of carvedilol added to the blank plasma was 94.2%. The detection limit was 3.6 ng/ml in the plasma. The reproducibilities (C.V.) were 2.7–7.5% for the within-day assay, and 2.6–7.4% for the between-day assay, indicating that the method was effective for the determination of carvedilol plasma levels.     

Quantification of carvedilol in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry: application to bioequivalence study

A rapid, sensitive and specific method to quantify carvedilol in human plasma using metoprolol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether solvent. After removed and dried the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile-water (50/50; v/v). The extracts were analyzed by a high performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed isocratically on Alltech Prevail C18 5 microm analytical column, (150 mm x 4.6 mm i.d.). The method had a chromatographic run time of 3.5 min and a linear calibration curve over the range 0.1-200 ng ml(-1) (r2>0.997992). The limit of quantification was 0.1 ng ml(-1). This HPLC-MS/MS procedure was used to assess the bioequivalence of two carvedilol 25 mg tablet formulations (carvedilol test formulation from Laboratórios Biosintética Ltda and Coreg from Roche Químicos e Farmacêuticos S.A standard reference formulation). A single 25 mg dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 2-week wash-out interval. Since the 90% CI for C(max) and AUCs ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration Agency, it was concluded that carvedilol formulation elaborated by Laboratórios Biosintética Ltda is bioequivalent to Coreg formulation for both the rate and the extent of absorption.     

Quantification of angiotensin-converting-enzyme-mediated degradation of human chemerin 145-154 in plasma by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

Chemerin is a chemoattractive protein acting as a ligand for the G-protein-coupled receptor ChemR23/CMKLR1 and plays an important role in the innate and adaptive immunity. Proteolytic processing of its C terminus is essential for receptor binding and physiological activity. Therefore, we investigated the plasma stability of the decapeptide chemerin 145-154 (P(145)-F(154)) corresponding to the C terminus of the physiologically active chemerin variant E(21)-F(154) from human hemofiltrate. For monitoring concentration-time profiles and degradation products we developed a novel matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry procedure using an internal peptide standard (hemorphin LVV-H7) for quantification. The linear range covers 2.5 orders of magnitude in the lower micromolar concentration range (lower limit of quantification 0.312 microg/ml, 0.25 microM) characterized by satisfactory reproducibility (CV < or =9%), accuracy (< or =10%), ruggedness, and recovery (98%). We found that chemerin 145-154 is C-terminally truncated in human citrate plasma by the cleavage of the penultimate dipeptidyl residue. N-terminal truncation was not observed. In contrast to citrate plasma, no degradation was detected in ethylenediammetetraacetate (EDTA) plasma. We identified angiotensin-converting-enzyme (ACE) to be responsible for C-terminal truncation, which could be completely inhibited by EDTA and captopril. These results are relevant to clarify the natural processing of chemerin and the potential involvement of ACE in mediating the immune response.     

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H(3)PO(4) with 0.2% triethylamine mobile phase was employed and separation was achieved with a C(18) analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9+/-0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 microg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 microg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 microg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 microg/ml. Results of the stability study indicated that when frozen at -80 degrees C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.     

Quantification of zolmitriptan in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of zolmitriptan in human plasma. After the addition of the internal standard (IS) and 1.0 M sodium hydroxide solution, plasma samples were extracted with methylene chloride:ethyl acetate mixture (20:80, v/v). The organic layer was evaporated under a stream of nitrogen at 40 degrees C. The residue was reconstituted with 100 microl mobile phase. The compounds were separated on a prepacked Lichrospher CN (5 microm, 150 mm x 2.0 mm) column using a mixture of methanol:water (10 mM NH(4)AC, pH 4.0) = 78:22 as mobile phase. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 0.30-16.0 ng/ml with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (R.S.D.%) were lower than 15% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.30 ng/ml. The proposed method enables the unambiguous identification and quantification of zolmitriptan for pharmacokinetic, bioavailability or bioequivalence studies.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

Determination of Cloretazine (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV相似文献   

Development and validation of a liquid chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and urine samples utilizing on-line sample preparation

Roscovitine, a purine analogue that selectively inhibits cyclin-dependent kinases, has been considered as a potential anti-tumor drug. The determination of roscovitine in plasma and urine was performed using microextraction in packed syringe as on-line sample preparation method with liquid chromatography and tandem mass spectrometry. The sampling sorbent utilized was polystyrene polymer. 2H3-lidocaine was used as internal standard. The limit of detection for roscovitine was as low as 0.5 ng/mL and the lower limit of quantification was 1.0 ng/mL. The accuracy and precision values of quality control samples were between +/-15% and < or =11%, respectively. The calibration curve was obtained within the concentration range 0.5-2000 ng/mL in both plasma and urine. The regression correlation coefficients for plasma and urine samples were > or =0.999 for all runs. The present method is miniaturized and fully automated and can be used for pharmacokinetic and pharmacodynamic studies.     

Ultra sensitive quantitation of endogenous oxytocin in rat and human plasma using a two-dimensional liquid chromatography-tandem mass spectrometry assay

Oxytocin (OT) is a neuropeptide with an extremely low endogenous level (low pg/ml) in human plasma. It is very challenging to develop a highly sensitive assay to measure endogenous OT, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) can provide high-throughput and selective methods for quantification of peptides in biological samples. A novel and highly sensitive two-dimensional LC–MS/MS (2D-LC–MS/MS) assay combining solid-phase extraction (SPE) has been developed and validated for the determination of endogenous OT in both human and rat plasma. The lower limit of quantification (LLOQ) was 1.00 pg/ml for human and 50.0 pg/ml for rat. Human plasma diluted with water (1:6, v/v) was successfully optimized as a surrogate matrix for human to prepare standard curves without endogenous interference. The extraction efficiency and absolute recovery were above 65.8% using the HLB SPE procedure, and matrix effects were lower than 12%. The method was validated in the range of 1.00–250 pg/ml for human plasma and 50.0–10,000 pg/ml for rat plasma with precision less than 12.7% and accuracy less than 7%.     

Separation of carvedilol enantiomers in very small volumes of human plasma by capillary electrophoresis with laser-induced fluorescence

A sensitive capillary electrophoretic method for the determination of carvedilol enantiomers in 100 μl of human plasma has been developed and validated. Carvedilol and the internal standard carazolol are isolated from plasma samples by liquid–liquid extraction using diethylether. A sensitive and selective detection is provided by helium–cadmium laser-induced fluorescence. The total analysis time is 17.5 min, about 30 min are needed for the sample preparation. The linearity of the assay ranges from 1.56 to 50 ng/ml per carvedilol enantiomer. The limits of quantification (LOQ) for the carvedilol enantiomers in 100 μl of human plasma are 1.56 ng/ml. The inter-day accuracy for R-carvedilol is between 95.8 and 103% (104% at LOQ) and for S-carvedilol between 97.1 and 103% (107% at LOQ); the inter-day precision values are between 3.81 and 8.64% (10.9% at LOQ) and between 5.47 and 7.86% (7.91% at LOQ) for R- and S-carvedilol, respectively. The small sample volume needed is especially advantageous for the application in clinical studies in pediatric patients. As an application of the assay concentration/time profiles of the carvedilol enantiomers in a 5-year-old patient receiving a test dose of 0.09 mg/kg carvedilol are reported.     

Headspace solid-phase microextraction and capillary gas chromatographic-mass spectrometric determination of rivastigmine in canine plasma samples

A simple, rapid and sensitive method for determination of rivastigmine in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC-MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 ml of plasma modified with 1.0 ml of sodium hydroxide-sodium carbonate solution (0.7 M:0.5M); extraction temperature of 100 degrees C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range from 0.2 to 80 ng/ml with regression coefficient corresponding to 0.9965 and coefficient of the variation of the points of the calibration curve lower than 10%. The quantification limit for rivastigmine in plasma was 0.2 ng/ml. The method was applied to determination of rivastigmine in canine plasma samples from animals after a single oral administration.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Simple and rapid docetaxel assay in plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and low cost sample preparation method was developed for quantification of docetaxel in mouse plasma by high-performance liquid chromatography/tandem mass spectrometry with paclitaxel as the internal standard. A small volume of plasma (40 microl) and one-step protein precipitation using methanol and acetonitrile (1:1 (v/v)) were used for sample preparation. The calibration curve for docetaxel in mouse plasma was linear over the range 25-2500 nM. The detection limit was 8 nM. The lower limit of quantitation is 25 nM. The intra- and inter-day precisions (CV) of analysis were 9.5 and 9.7% for the low quality control (LQC), 5.5 and 4.9% for the medium quality control (MQC) and 3.9 and 6.3% for the high quality control (HQC), respectively. The accuracy was 102.5% for LQC, 97.9% for MQC and 108.8% for HQC. This assay has now been applied to evaluation of mouse pharmacogenetics and other clinical pharmacology applications.     

Enantioselective and highly sensitive determination of carvedilol in human plasma and whole blood after administration of the racemate using normal-phase high-performance liquid chromatography

A highly sensitive HPLC method for enantioselective determination of carvedilol in human whole blood and plasma was developed. Carvedilol and S-carazolol as an internal standard extracted from whole blood or plasma were separated using an enantioselective separation column (Chiralpak AD column; 2.0 diameter x 250 mm) without any chiral derivatizations. The mobile phase was hexane:isopropanol:diethylamine (78:22:1, v/v). The excitation and emission wavelengths were set at 284 and 343 nm, respectively. The limits of quantification for the S(-)- and R(+)-carvedilol enantiomers in plasma and blood were both 0.5 ng/ml. Intra- and inter-day variations were less than 5.9%. As an application of the assay, concentrations of carvedilol enantiomer in plasma and blood samples from 15 patients treated with carvedilol for congestive heart failure were determined.     

Determination of schizandrin in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.     

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography-tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of the analgesic aminophenol ketobemidone in human plasma is presented. Two preparation methods for plasma samples containing ketobemidone were compared, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Both methods showed good precision (n=10), 1.7% and 2.9%, respectively (0.04 micro M) and 1.1% and 2.5%, respectively (0.14 micro M). The accuracy was 98% and 103%, respectively (0.04 micro M) and 105% and 99%, respectively (0.14 micro M). Ketobemidone could be quantified at 0.43 nM, with a relative standard deviation of 17.5% (n=19) using LLE and 18.6% (n=10) using SPE. This level was an order of magnitude lower than earlier reported quantification limits. Quantitative data from plasma samples analyzed with LC-MS-MS were in good agreement with those obtained by gas chromatography with chemical ionization mass spectrometry (GC-CI/MS). This indicates that LC-MS-MS is a good alternative method to GC-MS as it is more sensitive and time-consuming derivatization can be avoided.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.