Pharmacokinetic study of orphenadrine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

We developed and validated a simple, rapid, and accurate HPLC-MS/MS method with simple protein precipitation for the determination of orphenadrine. Injection-to-injection running time was 3 min with a retention time of orphenadrine of 1.1 min. The linear assay range was 1-200 ng/mL (r2 > 0.99). The intra- and inter-assay imprecisions were CV 0.6-4.2% and CV 1.6-6.1%, respectively. The accuracy, extraction recovery, specificity and stability were satisfactory. Using the measured plasma concentrations of orphenadrine in 24 healthy subjects, pharmacokinetic profiles of orphenadrine were evaluated (AUC(0-72,) 1565+/-731 ng h/mL, Cmax 82.8+/-26.2 ng/mL, Tmax 3.0+/-0.9 h, elimination half-life 25.8+/-10.3 h).     

Determination of ecabet in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a simple, robust and cost-effective assay for the determination of ecabet in human plasma. After a simple step of protein precipitation using methanol, plasma samples were analyzed by reverse phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) with valsartan as the internal standard (I.S.). Ecabet and the I.S. valsartan were separated on a Venusil MP C18 analytical column using methanol-10mM ammonium acetate (75:25, v/v, pH 3.0) as mobile phase at a flow rate of 1.0 mL/min. Ecabet and I.S. were eluted at 0.91 and 0.92 min, respectively, ionized in negative mode, and then detected by multiple reaction monitoring (MRM) essay. The MRM transitions of m/z 379.1-->m/z 277.1 and m/z 434.3-->m/z 350.1 were used to quantify ecabet and I.S., respectively. The assay was linear over the concentration range of 10-6000 ng/mL and was successfully applied to a pharmacokinetic study in healthy volunteers.     

Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.     

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

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 muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry

A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25-2500 ppm for MUS and 40-2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Amanita mushrooms naturally grown and circulated in the drug market.     

Determination of aldosterone in serum by liquid chromatography-tandem mass spectrometry

Measurement of serum aldosterone is clinically important in the diagnosis of hypertension. While isotope dilution gas chromatography-mass spectrometry (ID-GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. Detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. The analysis utilizes 0.5mL of serum. The samples were extracted with dichloromethane-ether. The extract was evaporated to dryness and aldosterone was analyzed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Aldosterone was also measured by RIA. The calibration curves for analysis of serum aldosterone exhibited consistent linearity and reproducibility in the range of 60-3000pmol/L. Interassay CVs were 4.3-7.5% at aldosterone concentrations of 97-993pmol/L. The lower limit of quantitation (LOQ) was 30pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to serum ranged from 95 to 102%. The regression equation by LC-MS/MS (x) and RIA (y) method was: y=1.33x+185 (r=0.95; n=124). Sensitivity and specificity of the LC-MS/MS method for serum aldosterone offer advantages over GC-MS by eliminating derivatization. The novel method is rapid, reliable and simple to perform with a routine LC-MS/MS spectrometer. The sensitivity is adequate for patient samples. Aldosterone concentrations reported by nonextraction RIA were consistently higher than those produced by LC-MS/MS.     

Determination of ambroxol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI)

A rapid, sensitive and specific method to determination of ambroxol in human plasma using high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/ESI) was described. Ambroxol and the internal standard (I.S.), fentanyl, were extracted from plasma by N-hexane-diethyl ether (1:1, v/v) after alkalinized with ammonia water. A centrifuged upper layer was then evaporated and reconstituted with 100 microl mobile phase. Chromatographic separation was performed on a BDS HYPERSIL C18 column (250 mmx4.6 mm, 5.0 microm, Thermo electron corporation, USA) with the mobile phase consisting of 30 mM ammonium acetate (0.4% formic acid)-acetonitrile (64:36, v/v) at a flow-rate of 1.2 mL min(-1). The total run time was 5.8 min for each sample. Detection and quantitation was performed by the mass spectrometer using selected ion monitoring at m/z 261.9, 263.8 and 265.9 for ambroxol and m/z 337.3 for fentanyl. The calibration curve was linear within the concentration range of 1.0-100.0 ng mL(-1) (r=0.9996). The limit of quantification was 1.0 ng mL(-1). The extraction recovery was above 83.3%. The methodology recovery was higher than 93.8%. The intra- and inter-day precisions were less than 6.0%. The method is accurate, sensitive and simple for the study of the pharmacokinetics and metabolism of ambroxol.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Determination of dioscin in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm x 4.6 mm i.d.) was packed with Hypersil ODS material (5 microm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.     

Determination of protodioscin in rat plasma by liquid chromatography-tandem mass spectrometry

Protodioscin (3-O-[alpha-L-rhamnopyranosyl-(1-->2)-{alpha-L-rhamnopyranosyl-(1-->4)}-beta-D-glucopyranosyl]-26-O-[beta-D-glucopyranosyl]-(25 R)-furost-5-ene-3 beta,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile-water-formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20-125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.     

Determination of Tirofiban in human serum by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method with a rapid and simple sample preparation was developed and validated for the determination of Tirofiban in biological fluids. Tirofiban in serum samples was extracted and cleaned up by using an automated solid phase extraction method. An external calibration was used. The mass spectrometer was operated in the multiple reaction monitoring mode (MRM). A good linear response over the range of 2-200ng/ml was demonstrated. The accuracy for Tirofiban ranged from 94.8 to 110.8% within-day and from 103.0 to 104.7% between-day. The lower limit of quantification was 2ng/ml. This method is suitable for pharmacokinetic studies.     

Determination of derivatized l-alanosine in plasma by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantitation of the cytotoxic antibiotic l-alanosine in human plasma. Alanosine was extracted from plasma by anion-exchange solid phase extraction, derivatized with dansyl chloride and analyzed by liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization in negative mode. Dansylation led to 50-fold improvement of method sensitivity over non-dansylated alanosine with a resulting 20 ng/ml limit of alanosine quantitation in plasma being achieved. The method was validated and applied for clinical studies of alanosine administered to cancer patients.     

Determination of olanzapine in human blood by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay was developed and validated to quantitatively determine olanzapine (OLZ) concentrations in human blood. Liquid-liquid extraction, using n-butanol:cyclohexane (3:47, v/v), was used to isolate OLZ and its internal standard, LY170158, from the biological matrix. Chromatographic resolution of OLZ from endogenous interferences and known metabolites was accomplished with a MetaChem Monochrom HPLC column (4.6 x 150 mm, d(p) 5 microm). Detection occurred using a Perkin-Elmer Sciex API III Plus triple quadrupole mass spectrometer using positive ion APCI and multiple reaction monitoring (MRM). The linear dynamic range was from 5 to 500 ng ml(-1) based on a 0.25-ml aliquot of human blood. The inter-day precision (%RSD) and accuracy (%RE) ranged from 3.65 to 10.64 and from -2.14 to 3.07, respectively. Modifications to an existing assay for the determination of OLZ in human plasma were necessary. A different structural analog was used as the internal standard due to instability observed for the original analog when using human blood as the matrix. A second modification was the addition of the anti-oxidant sodium ascorbate to inhibit degradation of OLZ in human blood, as has been noted by other investigators. Upon fortification of human blood with sodium ascorbate (final concentration, 0.33 mM), OLZ was found to be stable for at least 1 week at -70 degrees C as well as through two freeze-thaw cycles. This assay, which will be used to investigate the distribution of OLZ in human blood, grants insight into the proper sample handling conditions needed to perform valid determinations of OLZ in human blood.