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.     

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.     

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.     

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 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 benidipine in human plasma using liquid chromatography-tandem mass spectrometry

Fumonisins are water soluble mycotoxins produced by the fungus Fusarium verticillioides (formerly F. moniliforme). Fumonisin B(1) (FB(1)) is a diester of propane-1,2,3-tricarboxylic acid and 2-amino-12, 16-dimethyl-3,5,10,14,15-pentahydroxyeicosane, and is the most abundant of the naturally occurring fumonisins. Upon removal of the two tricarballylic acid side chains, the structure is referred to as hydrolyzed FB(1) (HFB(1)). FB(1) and HFB(1) are structurally similar to sphinganine, a sphingoid base. The fumonisins do not absorb UV light or fluoresce; therefore, derivatizing reagents are used for detection when separation is by high performance liquid chromatography (HPLC). The standard derivatizing reagent used for HPLC is ortho-phthalaldehyde (OPA) plus 2-mercaptoethanol (ME) reaction partner, however, the OPA-FB(1) derivative is not stable at room temperature. The objectives of this study were to: (1). determine the effect of temperature on the stability of the OPA-FB(1) derivative and (2). determine which structural characteristics of FB(1) contribute to the instability of the OPA-FB(1) derivative. The results indicate that OPA-FB(1), OPA-FB(3) and OPA-HFB(1) derivatives are unstable at 24 degrees C but that their stability improves significantly at 4 degrees C. The OPA-sphinganine derivative is stable for at least 24h at 24 degrees C. Thus, the instability of the OPA-FB(1) derivative may be attributed to its lack of a hydroxyl group at the carbon 1 position.     

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.     

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 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 etoricoxib in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionisation

The validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C(18) column (30x2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2-->280.2 and m/z 189.0-->104.1. The analytical method was validated over the concentration range 0.2-200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.     

Determination of meloxicam in human plasma by liquid chromatography-tandem mass spectrometry following transdermal administration

A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine meloxicam of low concentration in human plasma. After a simple sample preparation procedure by one-step protein precipitation with methanol, meloxicam and the internal standard piroxicam were chromatographed on a Zorbax SB C(18) column. The mobile phase consisted of acetonitrile-water-formic acid (80:20:0.2, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method had a lower limit of quantification of 0.10 ng/ml. The calibration curve was demonstrated to be linear over the concentration range of 0.10-50.0 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were within +/-2.5%), precise (intra- and inter-day relative standard deviation (R.S.D.) <7%). The validated method was successfully applied to the determination of meloxicam in human plasma collected up to 180 h after a transdermal administration of 30 mg meloxicam for evaluation of the pharmacokinetics.     

Determination of andrographolide in human plasma by high-performance liquid chromatography/mass spectrometry

In this paper, a rapid method based on high-performance liquid chromatography/electrospray-mass spectrometry (HPLC/ESI-MS) method for the quantitative determination of andrographolide (AND) in human plasma has been developed and validated. A liquid-liquid extraction (LLE) procedure was selected to isolate AND from biological matrixes. Isosorbide-5-mononitrate (IS-5-MN) was selected as the internal standard (IS). The correlation coefficient of the calibration curve was 0.998, in the range of 9.9-320.0 ng/mL. The validated method may be used to assess the bioavailability and pharmacokinetics of the drug.     

Rapid determination of tafenoquine in small volume human plasma samples by high-performance liquid chromatography-tandem mass spectrometry

A method was developed for the determination of tafenoquine (I) in human plasma using high-performance liquid chromatography-tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with methanol containing [2H3(15N)]tafenoquine (II) to act as an internal standard. The supernatant was injected onto a Genesis-C18 column without any further clean-up. The mass spectrometer was operated in the positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring mode. The assay required 50 microl of plasma and was precise and accurate within the range 2 to 500 ng/ml. The average within-run and between-run relative standard deviations were < 7% at 2 ng/ml and greater concentrations. The average accuracy of validation standards was generally within +/- 4% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze-thaw cycles and samples can safely be stored for at least 8 months at approximately -70 degrees C. The method was very robust and has been successfully applied to the analysis of clinical samples from patients and healthy volunteers dosed with I.     

Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C(18) column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350-->m/z 248 for ulifloxacin and m/z 362-->m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025-5.0 microg/ml for ulifloxacin with a lower limit of quantitation of 0.025 microg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 microg/ml. Compared with the reported LC method, the present LC-MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

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 p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C₁₈ column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2 → 120.2 and 152.1 → 110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1–500 μg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.