Determination of midazolam and 1'-hydroxymidazolam by liquid chromatography-mass spectrometry in plasma of patients undergoing methadone maintenance treatment

A rapid, sensitive and selective LC-MS method was developed for the simultaneous determination of midazolam (MDZ) and 1'-hydroxymidazolam (1'-OHMDZ) in plasma taken from 54 patients undergoing methadone maintenance therapy, most of whom were multidrug users. Samples spiked with prazepam, the internal standard, and were extracted into diethyl ether. Compounds were separated on a Phenomenex Luna C(18) column and a mobile phase of acetonitrile-ammonium acetate buffer (10 mM, pH 4.7) (52:48, v/v) at a flow-rate of 1 ml/min. The limit of detection was 0.65 and 0.68 (ng/ml) for MDZ and 1'-OHMDZ, respectively. Within-day relative standard deviations were less than 8%.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

Quantitative determination of erythromycylamine in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study of dirithromycin

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of erythromycylamine in human plasma was developed and validated. Erythromycylamine in plasma (0.2 mL) was extracted with ethyl acetate, the organic phase was transferred to another clear 1.5 mL Eppendorf tube and evaporated to dryness under gentle nitrogen stream at 45 degrees C, and the residue was dissolved in 100 microL of mobile phase. The samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150 mm x 2.1 mm I.D., 5 microm). A mobile phase containing 10 mM of ammonium acetate (pH = 6.4)-acetonitrile-methanol (50:10:40, v/v/v) was used isocratically eluting at a flow rate of 0.2 mL/min. Erythromycylamine and its internal standard (IS), midecamycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 4.5 to 720 ng/mL with r = 0.9997. The limit of quantification for erythromycylamine in plasma was 4.5 ng/mL with good accuracy and precision. The mean extraction recovery of the method was higher than 75.1% and 72.7% for erythromycylamine and IS, respectively. The intra-day and inter-day precision ranged from 5.2% to 6.4% and 5.6-9.3% (relative standard deviation, RSD), respectively. The established method has been successfully applied to a bioequivalence study of two dirithromycin formulations for 18 healthy volunteers.     

High-performance liquid chromatography-mass spectrometry method for determination of anagrelide in human plasma

This paper describes a simple, fast and sensitive liquid chromatography-mass spectrometry method for quantification of an anti-thrombocythemic agent, anagrelide in human plasma. The samples were subjected to a liquid-liquid extraction after addition of a buffer and an internal standard. Chromatography was performed on an Inertsil ODS2 column and the extract was injected onto a HPLC system coupled with mass spectrometric detection. Linear responses for standards were observed from 50 to 7500 pg/ml. The accuracy of intra-assay and inter-assay were in the ranges 4.3-4.4% and 4.8-5.6%, respectively. The method is simple and reproducible with a run time of less than 2 min.     

Sensitive liquid chromatography-tandem mass spectrometry method for the determination of cefixime in human plasma: application to a pharmacokinetic study

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine cefixime ((6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-(carboxymethoxyimino)acetamido]-8-oxo-3-vinyl-5-thia-1-azabicyclo-[4,2,0]-oct-2-ene-2-carboxylic acid) in human plasma. After a simple protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C(8) column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water-formic acid (40:60:0.5, v/v/v). The analyte and internal standard cefetamet were both detected by use of selected reaction monitoring mode. The method was linear in the concentration range of 0.05-8.0 microg/ml. The lower limit of quantification was 0.05 microg/ml. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 12.7%. The accuracy determined at three concentrations (0.05, 0.80 and 7.2 microg/ml for cefixime) was within +/-2.0% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method herein described was successfully applied for the evaluation of pharmacokinetic profiles of cefixime capsule in 24 healthy volunteers.     

High-performance liquid chromatography-mass spectrometry method for the determination of paroxetine in human plasma

A rapid and specific liquid chromatographic mass spectrometric (LC-MS-MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid-liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane-ethyl acetate. The standard curve was linear over a working range of 0.2-50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges -4.8 to -0.5% and -3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.     

Rapid and sensitive determination of vinorelbine in human plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C(18) column (50 mm×2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile-4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor→product ion transitions at m/z 779.4→122.0 and m/z 811.3→224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1-200 ng/mL (r>0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m(2) of vinorelbine to patients with metastatic breast cancer.     

High-performance liquid chromatography for the determination of 3-n-butylphthalide in rat plasma by tandem quadrupole mass spectrometry: application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 3-n-butylphthalide in rat plasma. Following protein precipitation with acetonitrile, 3-n-butylphthalide and glipizide (internal standard, I.S.) were separated using a gradient elution program on a C18 column and detected by mass spectrometry in positive ion mode with the multiple reaction monitoring (MRM) mode using the respective precursor to product ion combinations of m/z 191/145 for 3-n-butylphthalide and m/z 446/321 for glipizide, respectively. The total chromatographic running time was 2.5 min. The method was linear over the concentration range of 11.14-3480.00 ng/mL, using as little as 100 microL plasma. The lower limit of quantification (LLOQ) was 5.57 ng/mL. Finally, the method was successfully used to support a preclinical pharmacokinetic study of 3-n-butylphthalide in rats following intravenous administration.     

Simultaneous determination of oxymatrine and its active metabolite matrine in dog plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the quantification of oxymatrine (OMT) and its metabolite matrine (MT) in beagle dog plasma using a liquid-liquid extraction procedure followed by liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) analysis. Extend-C18 column (2.1 mm i.d. x 50 mm, 5 microm) with a C18 guard column (2.1 mm i.d. x 12.5 mm) was used as the analytical column. Linear detection responses were obtained for OMT concentration ranging from 5 to 4000 ng/ml and for MT concentration ranging from 5 to 2000 ng/ml. The precision and accuracy data, based on intra- and inter-day variations over 5 days, were lower than 5%. The limit of quantitation for OMT and MT were 2 and 1 ng/ml, respectively, and their recoveries were greater than 90%. Pharmacokinetic data of OMT and its active metabolite MT obtained with this method following a single oral dose of 300 mg OMT capsules to six beagle dogs was also reported for the first time.     

Simultaneous determination of dronedarone and its active metabolite debutyldronedarone in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

Dronedarone is a derivative of amiodarone--a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C(18) MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate-acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC-MS/MS method to a pharmacokinetic study after a single oral administration of 400mg dronedarone to six healthy volunteers.     

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.     

Quantitative determination of azithromycin in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of azithromycin in human plasma and its application in a pharmacokinetic study. With roxithromycin as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The analysis was carried out on an ACQUITY UPLC BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with gradient elution at flow rate of 0.35 mL/min. The mobile phase was 50 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 1-1000 ng/mL, with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was -1.3% to 5.7% at all QC levels. The method was applicable to clinical pharmacokinetic study of azithromycin in healthy volunteers following oral administration.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Determination of zolmitriptan in human plasma by liquid chromatography-tandem mass spectrometry method: application to a pharmacokinetic study

A sensitive and selective liquid chromatography-tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range 0.05-30 ng/ml with 0.5 ml of plasma using diphenhydramine as the internal standard. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring (SRM) mode using the atmospheric pressure chemical ionization (APCI) technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitrile-water-formic acid (70:30:0.5), at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intra-day precision (%R.S.D.) were less than 8.5% and accuracy (%error) was less than -2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 20 healthy volunteers.     

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.     

Enantioselective determination of lercanidipine in human plasma for pharmacokinetic studies by normal-phase liquid chromatography-tandem mass spectrometry

We describe here the first method for the enantioselective analysis of the calcium antagonist lercanidipine in human plasma by high performance liquid chromatography (HPLC) employing tandem mass spectrometric (MS) detection. Routine determination of lercanidipine enantiomers in human plasma in the working range of 0.025-50.0 ng ml(-1) plasma for each enantiomer with an accuracy and precision less than 15% was possible. Application of the method to a stereospecific study of the pharmacokinetics showed that plasma levels after an oral dose of rac-lercanidipine administered to a healthy volunteer were found to be higher for the (S)-enantiomer.     

Selective and rapid liquid chromatography-mass spectrometry method for the determination of lercanidipine in human plasma

A specific liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed and validated for the determination of lercanidipine, a dihydropyridine calcium channel blocker, in human plasma. Lercanidipine R-D3 was used as internal standard (IS). The drug was extracted from plasma using liquid-liquid extraction technique utilizing hexane: ethyl acetate as extraction solvent. The samples were analyzed using a prepacked Thermo Hypersil C(8) column and a mobile phase composed of a mixture of aqueous acetic acid and triethylamine in methanol. An ion trap mass spectrometer equipped with electrospray ionization (ESI) source operating in the positive ion mode was used to develop and validate the method. The method was proved to be sensitive and specific by testing six different human plasma batches. Linearity was established for the concentration ranges of 0.1-16 ng/ml with a regression factor of 0.9996. The lower limit of quantitation was identifiable and reproducible at 0.1 ng/ml with a precision of 7.2%.     

Determination of 25-OH-PPD in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-dammarane-3beta,12beta,20,25-tetrol (25-OH-PPD) in rat. Ginsenoside Rh(2) was employed as an internal standard. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. The mobile phase consisted of methanol-acetonitrile-10 mmol/l aqueous ammonium acetate (42.5:42.5:15, v:v:v), which was pumped at 0.4 ml/min. The analytical column (50 mm x 2.1 mm i.d.) was packed with Venusil XBP C8 material (3.5 microm). The standard curve was linear from 10 to 3000 ng/ml. The assay was specific, accurate (accuracy between -1.19 and 2.57% for all quality control samples), precise and reproducible (within- and between-day precisions measured as relative standard deviation were <5% and <7%, respectively). 25-OH-PPD in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 6h. The method had a lower limit of quantitation of 10 ng/ml, which offered a satisfactory sensitivity for the determination of (25-OH-PPD) in plasma. This quantitation method was successfully applied to pharmacokinetic studies of 25-OH-PPD after both an oral and an intravenous administration to rats and the absolute bioavailability is 64.8+/-14.3%.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.