Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

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 apolipoprotein A-I mimetic peptide D-4F in rabbit plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

The apolipoprotein A-I mimetic peptide D-4F is a potential therapeutical agent effective in maintaining cardiovascular health. A bioanalytical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) to quantitate the D-4F amount in rabbit plasma was developed and validated. A compound with a close structure similarity to the D-4F (only one amino acid A–V altered) was used as an internal standard. Both D-4F and the internal standard were extracted by protein precipitation using acetonitrile/0.2% Triton XL 80N. The correlation coefficient of the calibration curve was 0.9991 in the range 20–40,000 ng/mL. This assay can be used for pharmacokinetic studies of the drug. Also, it may be adjusted for the quantification of other members of apolipoprotein A-I mimetic peptide family.     

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray^™ (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-μl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 μl of plasma extracts onto a 100×3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y²). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and −0.2%, exhibiting precisions (C.V.) of ±6.5 and ±7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Simultaneous determination of five toxic alkaloids in body fluids by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel analytical method was developed and validated for the rapid and simultaneous analysis of five toxic alkaloids: Brucine, Strychnine, Ephedrine, Aconitine and Colchicine, in blood and urine using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (HPLC-ESI-MRM) mode. The linear range was 0.05-50.0 ng mL(-1) for Brucine, 0.1-50.0 ng mL(-1) for Strychnine and Ephedrine, 0.01-10.0 ng mL(-1) for Aconitine and Colchicine. The limits of quantification for Brucine, Strychnine, Ephedrine, Aconitine and Colchicine were found to be 0.03, 0.05, 0.20, 0.05, 0.01 ng mL(-1), respectively. The average extraction recoveries in urine ranged from 96.0 to 114.0% and in whole blood were 94.0 to 113.0%. The intra-day and inter-day RSDs were less than 8.3 and 10.6%, respectively. The five alkaloids could be well separated within 7 min in a single run. The established method should be suitable for the determination of trace alkaloids in body fluids.     

Determination of soyasaponins Ba and Bb in human serum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS-MS) has been developed and validated for the determination of soyasaponins Ba and Bb in human serum using glycyrrhizin as internal standard (I.S.). Soyasaponins Ba and Bb were extracted from human serum by liquid-liquid extraction and cleaned up by C(18) solid-phase extraction (SPE), followed by separation on a C(18) reversed-phase column using acetonitrile/water containing 0.025% acetic acid as a mobile phase for gradient elution. Soyasaponins Ba and Bb, and I.S. were ionized by negative ion pneumatically assisted electrospray and detected by HPLC-MS-MS in the multiple-reaction monitoring (MRM) mode using precursor-->product ion combinations at m/z 958-->940, 942-->924 and 822-->351, respectively. The calibration curves were linear (r(2)>0.991) in the concentration range of 0.5-100.0 ng/mL, with lower limits of quantification of 0.5 and 0.3 ng/mL for soyasaponins Ba and Bb, respectively, in human serum. Intra-day and inter-day relative standard deviations (R.S.D.) were less than 7.9 and 11.3%, respectively. The mean recoveries of soyasaponins Ba and Bb ranged from 92 to 101% and from 85 to 94%, respectively.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry

The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC-MS-MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6>348.2 for FV enantiomers and 307.1>161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL(-1) plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol, Novartis, S?o Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (-)-3S,5R-fluvastatin enantiomer, with an AUC (-)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (-)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.     

Simultaneous quantification of the new HIV protease inhibitors atazanavir and tipranavir in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl plasma for atazanavir and 50 microl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50 mm x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05-10 microg/ml for atazanavir and 0.1-75 microg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within +/-7.3 and +/-7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC-MS/MS assay for the quantification of protease inhibitors.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.     

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.     

Quantifying tetrapeptide SS-20 in rat plasma using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Quantitative analysis of peptides in biological matrices remains a challenging task. This is due to the low dosage and the complexity of both the matrix and the analytical characteristics of peptides. SS-20 is a tetrapeptide compound developed for the treatment of Parkinson's disease. To investigate the pharmacokinetics of SS-20, a sensitive and rapid liquid chromatography coupled with mass spectrometry method was developed and validated. An aliquot of 50 μL plasma sample was extracted via solid phase extraction. The extracts were separated using a hydrophilic interaction liquid chromatography column, and were then detected with a triple quadrupole mass spectrometer using electrospray ionization in positive-ion mode and selected reaction monitoring. The use of a deuterium-labeled internal standard provided acceptable accuracy, precision, and matrix effect. The lower limit of quantification was 0.30 ng/mL. The linear range of the method was from 0.30 to 1000 ng/mL. The intraday and interday precisions were lower than 10.2% in terms of relative standard deviation, and the accuracy was within ±2.1% in terms of relative error. The validated LC-MS/MS method was successfully applied to a pharmacokinetic study of SS-20 following an intravenous or subcutaneous injection administration of 1.0mg/kg to Sprague-Dawley rats.     

Quantitative determination of salidroside in rat plasma by on-line solid-phase extraction integrated with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method for the quantification of salidroside, a major biologically active compound in Rhodiola, in rat plasma by on-line SPE LC/MS/MS in negative electrospray mode was developed and validated. A column-switching instrument and two HPLC pumping systems were employed, and salicin was used as the internal standard. A Waters Oasis HLB extraction column and an Agilent TC-C(18) analytical column in a column-switching set-up with gradient elution were utilized. The MS/MS ion transitions monitored were m/z 299.0/119.0 and 285.1/122.9 for salidroside and salicin, respectively. The standard curves were linear within a range of 50-5000 ng/mL using weighted linear regression analysis (1/x). The intra- and inter-day coefficients of variance ranged from 1% to 9%. The recovery was above 90%. The freeze/thaw and long-term stability were validated. This method was subsequently applied to a pharmacokinetic study of salidroside in rats.     

Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An accurate and reliable liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.). The analyte and the I.S. were extracted from acidified plasma by methyl-t-butyl ether. Separation was achieved on a YMCbasic column using acetonitrile-water-formic acid mobile phase. The MS-MS detection was by monitoring fragmentation 372.1-->326.2 (m/z) for COL-3 and 255.1-->153.1 (m/z) for the I.S. on a Sciex API 365 using a Turbo Ionspray in positive ion mode. The retention times were approximately 1.7 min for COL-3 and 1.8 min for the I.S. The validated dynamic range was 0.03-10.0 microg/ml using 0.25-ml plasma with correlation coefficients of >or=0.9985. The precision and accuracy for the calibration standards (n=3) were RSD相似文献   

Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

Quantitative determination of the diastereoisomers of hexabromocyclododecane in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A sensitive, simple and feasible method has been developed and validated for the simultaneous determination of three diastereoisomers of hexabromocyclododecane (HBCD) in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). The simple pretreatment generally involved protein precipitation with methanol (MeOH). The separation was performed with a C18 reverse phase column. The mobile phases were 5mM ammonium acetate (NH(4)AC) in water and acetonitrile (ACN). The mass spectrometer was operated using negative electrospray ionization (ESI) source and the data acquisition was carried out with multiple reaction monitoring (MRM) mode. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The method was partially validated with the evaluations of accuracy, precision, linearity, limit of quantification (LOQ), limit of detection (LOD), recovery, matrix effect and carryover effect. With the present method, the intra-batch accuracies were 94.7-104.3%, 91.9-109.3% and 89.8-105.0% for α-, β- and γ-HBCD, respectively. And the inter-batch accuracies were ranged from 94.2% to 109.7%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 11.2%. The recoveries were from 79.0% to 108.9% and the LOQ was 10pg/mL for each diastereoisomer. The linear range was 10-10,000pg/mL with the linear correlation coefficient R(2)>0.996. No significant matrix effect and carryover effect of the analytes were observed in this study. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of HBCDs in human plasma.