Development and validation of a high-performance liquid chromatography-electrospray ionization-mass spectrometry assay for the determination of zaleplon in human plasma

In this paper, a sensitive and rapid chromatographic procedure using a selective analytical detection method (electrospray ionization-mass spectrometry in selected-ion monitoring mode) in combination with a simple and efficient sample preparation step is first presented for the determination of zaleplon in human plasma. The separation of the analyte, internal standard, and possible endogenous compounds are accomplished on a phenomenex Luna 5-microm C8(2) column (250- x 4.6-mm i.d.) with methanol-water (75:25, v/v) as the mobile phase. In order to optimize the mass detection of zaleplon, several parameters such as ionization mode, fragmentor voltage, m/z ratios of ions monitored, type of organic modifier, and eluent additive in the mobile phase are discussed. An internal standard is selected to guarantee the quantitative accuracy. Each analysis takes less than 6 min. The calibration curve of zaleplon in the range of 0.1-60.0 ng/mL in plasma is linear with a correlation coefficient of > 0.9992, and the detection limit (s/n = 3) is 0.1 ng/mL. The within- and between-day variations (relative standard deviation) in the zaleplon plasma analysis are less than 2.4% (n = 15) and 4.7% (n = 15), respectively. The application of this method is demonstrated for the analysis of zeleplon plasma samples in a Phase-I human pharmacokinetic study.     

High-performance liquid chromatography-electrospray ionization mass spectrometric determination of nisoldipine in human plasma

A selective and sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (MS) for the determination of nisoldipine in human plasma is first presented. With nimodipine as the internal standard, nisoldipine is extracted from plasma with ethyl acetate. The organic layer is evaporated and the residue is resuspended in the mobile phase of methanol-water (80:20, v/v). An aliquot of 40 microL is chromatographically analyzed on an Agilent ODS C18 reversed-phase column (5 microm, 250- x 4.6-mm i.d.) by means of selected-ion monitoring mode MS. The calibration curve of nisoldipine in plasma exhibits a linear range from 0.5 to 20.0 ng/mL with a correlation coefficient of 0.9995. The limit of detection is 0.2 ng/mL. The within- and between-day variations (relative standard deviation) are less than 9.28% and 11.13% (n = 5), respectively. The developed method is validated through successful use for the analysis of nisoldipine contained in biological fluids resulting from a phase-I human pharmacokinetic study.     

Quantitative determination of medroxyprogesterone acetate in plasma by liquid chromatography/electrospray ion trap mass spectrometry.

A sensitive and rapid liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of medroxyprogesterone acetate (MPA) in human plasma. Plasma samples (1.0 mL) were simply extracted with pentane and the extracts were analyzed by HPLC with the detection of the analyte in the selective reaction monitoring (SRM) mode. The determination of MPA was accurate and reproducible, with a limit of quantitation of 0.05 ng/mL in plasma. The standard calibration curve for MPA was linear (r = 0.998) over the concentration range 0.05-6.0 ng/mL in human plasma. Analysis precision over the concentration range of MPA was lower than 18.8% (relative standard deviation, RSD) and accuracy was between 96.2 and 108.7%.     

A rapid and specific approach for direct measurement of donepezil concentration in human plasma by LC-MS/MS employing solid-phase extraction

A selective, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C(8), 5 microm, 100 x 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15-50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were < or =8.92 and 10.35% and accuracy (%RE) were < or =7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps.     

Quantification of doxazosin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry

Hydrophilic interaction LC with MS/MS (HILIC-MS/MS) was described as a rapid, sensitive, and selective method for the quantification of doxazosin in human plasma. Doxazosin and cisapride (internal standard) were extracted from human plasma with ethyl acetate at alkaline pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of ACN/ammonium formate (100 mM, pH 4.5) (93:7 v/v). The analytes were detected using an ESI MS/MS in the selective-reaction-monitoring mode. The standard curve was linear (r = 0.9994) over the concentration range of 0.2-50 ng/mL. The LOQ for doxazosin was 0.2 ng/mL using 100 microL plasma sample. The CV and relative error for intra- and interassay at four QC levels were 3.7-8.7% and 0.0-9.8%, respectively. The matrix effect for doxazosin and cisapride were practically absent. The recoveries of doxazosin and cisapride were 67.4 and 61.7%, respectively. This method was successfully applied to the pharmacokinetic study of doxazosin in humans.     

Target metabolic profiling analysis of free amino acids in plasma as EOC/TBDMS derivatives by GC-SIM-MS

Metabolic profiling analysis of free amino acids (AAs) in plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring mode after ethoxycarbonyl/tert-butyldimethylsilyl derivatives. Characteristic fragment ions, including [M - 57](+) ions, permitted sensitive and selective detection of most of the AAs in the presence of co-extracted carboxylic acids, including free fatty acids, at much higher levels. The overall method was linear (r > or = 0.9991), reproducible (relative standard deviation = 2.3-8.8%) and accurate (relative error = -7.3-7.7%) with detection limits of 0.01-1.9 ng/mL. A total of 18 AAs, 15 protein AAs and three nonprotein AAs were quantitatively screened in a normal human plasma sample. This selective and simple method using a minimal sample volume was effective for the quantitation of plasma free AAs.     

High-performance liquid chromatography-electrospray ionization-mass spectrometric determination of emedastine difumarate in human plasma and its pharmacokinetics

A selective and sensitive method employing high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry is developed and validated for the determination of emedastine difumarate in human plasma. With naphazoline hydrochloride as the internal standard, emedastine difumarate is extracted from plasma with ethyl acetate. The organic layer is evaporated, and the residue is redissolved in the mobile phase. An aliquot of 10 microL is chromatographically analyzed on a prepacked Phenomenex Luna 5u CN 100A (150 x 2.0-mm i.d.) column, using a mobile phase comprised of methanol-water (20 mM CH(3)COONH(4), pH 4.0) (80:20, v/v). Standard curves are linear (r(2) = 0.9990) over the concentration range of 0.05-30 ng/mL and had good accuracy and precision. The within- and between-batch precisions did not exceed 15% for the relative standard deviation. The lower limit of detection is 0.01 ng/mL. The validated HPLC-ESI-MS method is successfully used to study emedastine difumarate pharmacokinetics in 12 healthy volunteers.     

Simultaneous clinical monitoring of lactic acid, pyruvic acid and ketone bodies in plasma as methoxime/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry in selected ion monitoring mode

Simultaneous determination of lactic acid, pyruvic acid, 3-hydroxybutyric acid and acetoacetic acid for clinical monitoring of lactic acidosis and ketone body formation in human plasma (20 microL) was performed by gas chromatography-mass spectrometry in selected ion monitoring (SIM) mode after generating methoxime/tert-butyldimethylsilyl derivatives. All of the targeted carboxylic acids were detected by characteristic fragment ions, which permitted sensitive and selective identification in the presence of co-extracted free fatty acids and other acidic metabolites at much higher levels. The method was linear (r>or=0.9991), reproducible (% relative standard deviation=1.2-5.8), and accurate (% relative error=-7.2-7.6), with detection limits of 0.05-1.7 ng/mL. This rapid, accurate and selective method using minimal plasma samples (20 microL) is useful in the clinical monitoring of lactic acidosis and ketone body formation in plasma.     

Adsorptive stripping voltammetric assay of phenazopyridine hydrochloride in biological fluids and pharmaceutical preparations

A sensitive method for the measurement of phenazopyridine hydrochloride (PAP) by differential pulse polarography (DPP) based on adsorptive stripping technique, using a hanging mercury drop electrode (HMDE) is described. The voltammetric peak is obtained at -0.760 V, which corresponds to the reduction of the azo group in Britton-Robinson buffer. The redox behaviour is reversible. Optimum conditions were found to be: accumulation potential -50 mV (vs. Ag/AgCl), accumulation time 60 s, scan rate 5 mV s(-1), pulse amplitude -100 mV and supporting electrolyte Britton-Robinson buffer (0.04 M, pH=11). The relative standard deviation (at 20 ng ml(-1) level) was +/-0.6% for six measurements. The calculated detection limit was 0.0299 ng ml(-1) with a 60-s accumulation time. The applicability of such a method was evaluated through the assay of PAP in human plasma and urine samples after a simple extraction procedure and in pharmaceutical preparation. The mean recovery was 97+/-2 (100 ng ml(-1) plasma).     

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans

A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C(18) column and detected by ultraviolet absorbance at 239 nm. The use of methanol-water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a fl ow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0-300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4-104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 +/- 14.3 ng/mL at 3.1 +/- 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 +/- 83.1 ng.h/mL.     

A sensitive, rapid and specific determination of midazolam in human plasma and saliva by liquid chromatography/electrospray mass spectrometry

A rapid, sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ES-MS) method was developed for the quantitative determination of the anaesthetic benzodiazepine midazolam (MID) in human saliva and plasma from patients undergoing anesthesia procedures. Biological samples spiked with diazepam-d5, the internal standard, were extracted into diethyl ether. Compounds were separated on a Xterra RP18 column using a mobile phase of acetonitrile/formic acid 0.1% at a flow rate of 0.25 mL/min under a linear gradient. Column effluents were analyzed using MS with an ES source in the positive ionization mode. Calibration curves were linear in the concentration ranges of 1-250 and 0.2-25 ng/mL in plasma and saliva, respectively. The limits of detection were 0.5 ng/mL in plasma and 0.1 ng/mL in saliva, using a 0.5-mL sample volume. The recoveries of the spiked samples were above 65%. The method was applied to ten real samples from patients undergoing midazolam treatment.     

Liquid chromatography tandem mass spectrometry method for the quantification of amisulpride with LLOQ of 100 pg/mL using 100 microL of plasma

A sensitive and selective high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of amisulpride in 100 microL of human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective (M + H)(+) ions, m/z 370-242 for amisulpride and m/z 341-112 for the internal standard. The assay exhibited a linear dynamic range with a lower range of 0.1-100 ng/mL and a higher range of 1-500 ng/mL of amisulpride in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for both linearity ranges. A run time of 2.0 min for each sample made it possible to analyze more than 275 human plasma samples per day. The validated method has been successfully used to analyze plasma samples for application in pharmacokinetic studies.     

High-performance liquid chromatographic assay of indomethacin in porcine plasma with applicability to human levels

A high-performance liquid chromatography (HPLC) assay is described for the determination of indomethacin in porcine plasma using acetonitrile to precipitate plasma proteins and for the one-step extraction. Calibration curves (using the internal standard method) are linear (r2 > 0.98) over the concentration range of 50.0 to 3000 ng/mL in both mobile phase and plasma. Precision, expressed as the inter- and intraday coefficient of variation (n = 5), is < 7% on the same day and < 5% between days at each plasma control sample of 300, 1000, and 3000 ng/mL, respectively. System precision, calculated as the coefficient of variation (n = 5), is < 7% at 3000 ng/mL of indomethacin, and the limit of quantitation in plasma is 50 ng/mL. The absolute recovery for both indomethacin and the internal standard (mefenamic acid) from plasma is over 97% (n = 3), and the concentrations do not deviate more than -2.9% to 2.4% from their actual values. The specificity of the method is confirmed. This technique is thus reported to be both rapid and specific. The real advantage is the small sample volume required (500 microL), which allows it to be considered for the quantitation of indomethacin in plasma from paediatric patients.     

Determination of midazolam in human plasma by solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of midazolam, a short-acting 1,4-imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid-phase microextraction (SPME) of midazolam using an 85-microm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5-300 ng/mL, relative intra- and inter-assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.     

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

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS) quantitative detection method, using cefalexin as internal standard, was developed for the analysis of faropenem in human plasma and urine. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C18 reversed-phase column with 0.1% formic acid-methanol (45:55, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves with good linearities (r=0.9991 for plasma sample and r=0.9993 for urine sample) were obtained in the range 5-4000 ng/mL for faropenem. The limit of detection was 5 ng/mL. 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 faropenem in humans, and to our knowledge, it is the first time the pharmacokinetic of faropenem has been elucidated in vivo using LC-MS/MS.     

Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high-performance liquid chromatography and fluorescence detection

A validated new and precise reversed-phase high-performance liquid chromatographic method for the determination of melatonin in human plasma and cerebrospinal fluid, with 5-fluorotryptamine as internal standard, is described. Liquid-liquid extraction with dichloromethane was performed under alkaline conditions. After evaporation of the organic solvent, the extract was dissolved in eluent and chromatographed on a base-deactivated octadecyl column, using an eluent composed of 650 mL potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 350 mL methanol. Fluorescence detection at an excitation wavelength of 224 nm and an emission wavelength of 348 nm was used for quantitation. Melatonin and 5-fluorotryptamine chromatographed with retention times of 5.3 and 9. 3 min, respectively. Mean recoveries of 96% (n = 10) and 95% (n = 5) were found for melatonin in plasma and cerebrospinal fluid respectively. 5-Fluorotryptamine was found to have a mean recovery of 90% (n = 10) and 82% (n = 5) in plasma and cerebrospinal fluid, respectively. The repeatability coefficients of variation for both melatonin and 5-fluorotryptamine in plasma were 4-5% [five different samples (r = 5) on two consecutive days (n = 2)], with reproducibility coefficients of 1.6-7% (n = 2, r = 5) and 0.9-4% (n = 2, r = 5) for melatonin and internal standard, respectively. In cerebrospinal fluid the repeatability coefficient of variation of the extraction procedure was 5% (n = 1, r = 5) for melatonin and 7% (n = 1, r = 5) for 5-fluorotryptamine. The correlation coefficients of the calibration curves were 0.9998 (n = 2) in plasma at a concentration range of 0.108-25.9 ng/mL and 0.9994 (n = 2) at a concentration range of 0.108-25.9 ng/mL in cerebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

Determination of perfluorooctane sulfonate and perfluorooctanoic acid in human plasma by large volume injection capillary column switching liquid chromatography coupled to electrospray ionization mass spectrometry

Rapid, selective, and sensitive methodology for the quantification of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in human plasma using packed capillary liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry has been developed. Plasma proteins were precipitated using acetonitrile and the resulting supernatant was diluted 1+1 with water containing 10 mM ammonium acetate (NH4Ac) prior to injection. Sample volumes of 250 microL were loaded onto a 30 mm x 0.32 mm ID 10 microm Kromasil C18 precolumn by a carrier solution consisting of 10 mM NH4Ac in ACN/H2O (5/95, v/v) at a flow rate of 100 microL/min, providing on-line analyte enrichment and sample clean-up. Backflushed elution onto a 100 mm x 0.32 mm ID 3.5 microm Kromasil C18 analytical column was conducted using an ACN/H2O solvent gradient containing 10 mM NH4Ac. In order to improve the robustness and performance of the method, perfluoroheptanoic acid (PFHA) was used as internal standard. Separation and detection of PFOA, PFHA, and PFOS were achieved within 10 minutes. Ionization was performed in the negative mode in the m/z range 250-550. The method was validated over the concentration range 1-200 ng/mL for PFOA and over the range 5-200 ng/mL untreated plasma for PFOS, yielding correlation coefficients of 0.997 (PFOA) and 0.996 (PFOS), respectively. The within-assay (n = 6) and between-assay (n = 6) precisions were in the range 2.1-9.2 and 5.6-12%, respectively. The concentration limits of detection (cLOD) of PFOA was 0.5 ng/mL while the cLOD of PFOS was estimated to be 0.2 ng/mL in untreated plasma.     

Determination of huperzine A in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study on Chinese volunteers

A simple, sensitive and selective LC-MS-MS method has been developed for the quantification of huperzine A in human plasma. Huperzine A and pseudoephedrine hydrochloride (internal standard) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a C(18) column with a mobile phase consisting of 0.2% formic acid-methanol (15:85, v/v) and detected using a tandem mass spectrometer with an electrospray ionization interface. The lower limit of quantification was 0.0508 ng/mL, and the assay exhibited a linear range of 0.0508-5.08 ng/mL (r = 0.9998). The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test vs reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting means of major pharmacokinetic parameters such as AUC(0-24), AUC(0-infinity), C(max), T(max) and t(1/2) of huperzine A were 16.35 +/- 3.42 vs 16.38 +/- 3.61 ng h/mL, 17.53 +/- 3.80 vs 17.70 +/- 3.97 ng h/mL, 2.47 +/- 0.49 vs 2.51 +/- 0.51 ng/mL, 1.3 +/- 0.4 vs 1.2 +/- 0.3 h and 5.92 +/- 0.75 vs 6.18 +/- 0.66 h, respectively, indicating that these two kinds of tablets were bioequivalent.     

Quantitative analysis of cimetidine in human plasma using LC/APCI/SRM/MS.

A quantitative method was developed and validated for rapid and sensitive analysis of cimetidine in human plasma. The method involved the use of liquid chromatography (LC) coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry (MS). A cimetidine analog, SKF92374, was used as the internal standard. Separation of cimetidine and the internal standard was accomplished using a reverse-phase HPLC column (C18). The eluted components were ionized by the APCI source and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 5 ng/mL (lower limit of quantitation) to 10,000 ng/mL. The results demonstrated excellent precision (%RSD 1. 1-8.9%) and accuracy (94.7-108.0%) over this range. In addition, the amount of plasma sample needed for analysis was small (50 muL), and the plasma pretreatment (analyte recovery >94%) was simple and time saving. This assay was used to evaluate cimetidine levels in premature infants following intravenous infusion of cimetidine.