A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma

Artemisinin is a widely used antimalarial drug. To evaluate the pharmacokinetics of artemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of artemisinin in rat plasma. For detection, a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization (ESI) TurboIonSpray inlet in the positive ion multiple reaction monitoring (MRM) mode was used to monitor precursor ([M+NH4]+) --> product ions of m/z 300.4 --> 209.4 for artemisinin and m/z 316.4 --> 163.4 for artemether, the internal standard (IS). The plasma samples were pretreated by a simple liquid-liquid extraction with ether. The standard curve was linear (r > 0.99) over the artemisinin concentration range of 1.0-200.0 ng/mL in plasma. The method had a lower limit of quantification of 1.0 ng/mL for artemisinin in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of artemisinin. The intra- and inter-day precisions were measured to be within +/-5.3% and accuracy between -2.6% and 1.2% for all quality control samples, lower limit of quantification and upper limit of quantification samples. The extraction recoveries of artemisinin and the IS were 95.4 +/- 4.5% and 92.8 +/- 3.9%, respectively. This present method was successfully applied to the characterization of the pharmacokinetic profile of artemisinin in rats after oral administration.     

Sensitive sulphur-specific detection of omeprazole metabolites in rat urine by high-performance liquid chromatography/inductively coupled plasma mass spectrometry

The use of high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) with sulphur-specific detection was investigated as a method for obtaining metabolite profiles for the drug omeprazole administered as a 1:1 mixture of (32)S- and (34)S-labelled material. Analysis based on the monitoring of the chromatographic eluent at either m/z 32 or 34 was not successful due to insufficient sensitivity caused by interferences from polyatomic ions. However, reaction of sulphur with oxygen in the hexapole collision cell, combined with monitoring at m/z 48 (for (32)S) or m/z 50 (for (34)S), provided a facile method for metabolite profiling. Detection of m/z 48 was superior in sensitivity to detection of m/z 50.     

Quantitative determination of E5880 in rat plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A simple and sensitive method is described for the determination of E5880 in rat plasma. The method is based on high-performance liquid chromatography/electrospray ionization mass spectrometry, using deuterated E5880 as an internal standard. Selected reaction monitoring is employed for selectivity and sensitivity, this in turn enables quantification in a short period of time (within 7 minutes) over the extended range of 0.1-1000 ng/ml with acceptable precision and accuracy. The method demonstrated to be suitable for the quantitative analysis of E5880 in rat plasma. The pharmacokinetic profile of E5880 after a single intravenous administration of E5880 was elucidated.     

Sensitive liquid chromatography tandem mass spectrometry method for the quantification of Quetiapine in plasma

A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of quetiapine in rat plasma. Following liquid-liquid extraction, the analyte was separated using a gradient 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 384 to m/z 221 for quetiapine and m/z 327 to m/z 270 for the internal standard. The assay exhibited a linear dynamic range of 0.25-500 ng/mL for quetiapine in rat plasma. The lower limit of quantification was 0.25 ng/mL with a relative standard deviation of less than 7%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated method was successfully used to analyze rat plasma samples for application in pre-clinical pharmacokinetic studies. This method in rodent plasma could be adapted for quetiapine assay in human plasma.     

Sensitive liquid chromatography/tandem mass spectrometry method for the determination of two novel highly lipophilic anticancer drug candidates in rat plasma and tissues

A simple and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) method was developed and validated for determination of two highly lipophilic anticancer drug candidates, LG1980 and GH501, in rat plasma and tissues (liver, kidney and femur bones). LG1980 and GH501 were extracted from rat plasma and tissue homogenates using liquid–liquid extraction. The method provided a linear range of 1.0–200.0 ng/mL for GH501 in plasma and LG1980 in plasma and liver. For both analytes in other tissue homogenates the linear range was 2.0–400.0 ng/mL. The method was validated with precision within 15% relative standard deviation, accuracy within 15% relative error and a consistent recovery. This method has been successfully applied in two preclinical studies for LG1980 and GH501 to determine their concentrations in rat plasma, liver, kidney and bone over 24 h after intravenous injection of compounds.     

Quantitative determination of sparfloxacin in rat plasma by liquid chromatography/tandem mass spectrometry

Sparfloxacin, a fluoroquinolone antibiotic, is used for the treatment of bacterial infection. A quantification method using mass spectrometry was developed for the determination of sparfloxacin in rat plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed‐phase C₁₈ column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma sparfloxacin concentrations after a single oral administration of sparfloxacin in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

A liquid chromatography/tandem mass spectrometry method for determination of aristolochic acid‐I in rat plasma

A sensitive, rapid and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the determination of aristolochic acid‐I (AA‐I) in rat plasma. Finasteride was used as the internal standard (IS). The analyte was separated on a Zorbax Eclipse XDB‐C₁₈ column by isocratic elution with methanol‐10 mM ammonium acetate (75:25, v/v, pH = 7.3) at a flow rate of 0.25 mL/min, and analyzed by mass spectrometry in positive multiple reaction monitoring mode. The precursor‐to‐product ion transitions of m/z 359.0 → 298.2 and m/z 373.1 → 305.2 were used to detect AA‐I and IS, respectively. Good linearity was achieved over a range of 0.4–600 ng/mL. Intra‐ and inter‐day precisions measured as relative standard deviation were less than 13.5%, and accuracy ranged from 94.2 to 97.5%. The developed method was successfully applied in the pharmacokinetic study of AA‐I in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

A sensitive method for determination of salvianolic acid A in rat plasma using liquid chromatography/tandem mass spectrometry

Salvianolic acid A (SAA), a major effective constituent of Salvia miltiorrhizas, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated for SAA in rat plasma, which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA. The method used liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with chloramphenicol as the internal standard. A simple liquid-liquid extraction based on ethyl acetate was employed. The combination of a simple sample cleanup and short chromatographic run time (3 min) increased the throughput of the method substantially. The method was validated over the range 1.4-1000 ng/mL with a correlation coefficient >0.99. The lower limit of quantification was 1.4 ng/mL for SAA in plasma. Intra- and inter-day accuracies for SAA were 95-113 and 98-107%, and the inter-day precision was less than 12%. This method is more sensitive and faster than previous methods. After a single oral dose of 100 mg/kg of SAA, the mean peak plasma concentration (Cmax) of SAA was 318 ng/mL at 0.5 h, the area under the plasma concentration-time curve (AUC0-12 h) was 698 +/- 129 ng.h/mL, and the elimination half-life (T1/2) was 3.29 h.     

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100–20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.     

High-performance liquid chromatography/tandem mass spectrometry method for the determination of arbidol in human plasma

An HPLC/MS/MS method for the determination of arbidol in human plasma was developed. Arbidol and internal standard (loratadine) were extracted from alkaline plasma with tert-butyl methyl ether and analyzed on a Zorbax SB C18 column (30 x 2.1 mm id, 3.5 microm particle size). The detection was by monitoring arbidol at m/z 479.1 --> 434.1 and the internal standard at m/z 383.2 --> 337.2. The method was validated according to U.S. Food and Drug Administration guidelines. The calibration curve was linear over the range of 0.5-500 ng/mL using a 100 microL sample volume. The intraday and interday precisions were less than 6.5%, and acceptable values were obtained for accuracy, recovery, and sensitivity. The developed method was selective, simple, sensitive, and easily applicable.     

Ultra-performance liquid chromatography/tandem mass spectrometry method for the determination of lercanidipine in human plasma

A simple, sensitive and rapid ultra-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Lercanidipine and the internal standard, nicardipine, were extracted from plasma by liquid-liquid extraction using tert-butyl methyl ether as the extraction solvent. UPLC analysis was performed isocratically on an AcQuity UPLC BEH C18 analytical column (2.1 x 50.0 mm i.d., particle size 1.7 microm). The mobile phase consisted of 70% acetonitrile in water containing 0.2% v/v formic acid and pumped at a flow rate of 0.30 mL/min. ESI in positive ion mode, with multiple reaction monitoring (MRM), was chosen for the detection of the analytes. The assay was linear over a concentration range of 0.05-30 ng/mL for lercanidipine with a limit of quantitation of 0.05 ng/mL. Quality control samples (0.05, 0.15, 15 and 25 ng/mL) in five replicates from five of analytical runs demonstrated intra-assay precision (% CV < or =7.3%), inter-assay precision (% CV < or =6.1%) and an overall accuracy (% relative error) of less than 6.2%. A run time of less than 1.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to quantify lercanidipine in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Development of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of fenofibric acid in rat plasma

A rapid and sensitive LC‐MS/MS method for the quantification of fenofibric acid in rat plasma was developed and validated. Plasma samples were prepared by liquid–liquid extraction with a mixture of N‐hexane–dichloromethane–isopropanol (100:50:5, v/v/v). Isocratic chromatographic separation was performed on a reversed‐phase Discovery C₁₈ column (2.1 × 50 mm, 5 µm). The mobile phase was methanol–water–formic (75:25:0.25, v/v/v). Detection of fenofibric acid and the internal standard (IS) diclofenac acid was achieved by ESI MS/MS in the negative ion mode using m/z 317 → m/z 213 and m/z 294 → m/z 250 transitions, respectively. The method was linear from 0.005 to 1.250 µg/mL when 100 μL plasma was analyzed. The lower limit of quantification was 0.005 µg/mL. The intra‐ and inter‐day precision values were below 8.2%, and accuracy ranged from ?0.9 to 2.1% in all quality control samples. The recovery was 90.3–94.7% and 83.3% for fenofibric acid and IS, respectively. Total run time for each sample analysis was 2.5 min. The validated method was successfully applied to a pharmacokinetic study in six rats after oral administration of fenofibrate, the ester prodrug of fenofibric acid (equivalent to fenofibric acid 5 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid fenofibric acid determination. Copyright © 2011 John Wiley & Sons, Ltd.     

Sensitive liquid chromatography/mass spectrometry assay for the quantification of azithromycin in human plasma

A simple, rapid, sensitive and specific liquid chromatography/electrospray ionization mass spectrometry method was developed and validated to quantify azithromycin in human plasma, using erythromycin as the internal standard (IS). A simple sample preparation method of protein precipitation with methanol was employed. Methanol, acetonitrile and water (12:30:58, v/v/v) were used as the isocratic mobile phase, with 0.1% formic acid and 0.1% ammonium acetate in water. Selected ion monitoring was specific for azithromycin and erythromycin. The assay was linear over the concentration range 4.69-600 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9994 to 0.9998. The intra- and inter-day precisions, calculated from quality control samples, were less than 8.24%. The method was employed in a pharmacokinetic study after oral administration of 500 mg azithromycin dispersible tablet to 20 healthy volunteers.     

Quantitative determination of BMS-378806 in human plasma and urine by high-performance liquid chromatography/tandem mass spectrometry

BMS-378806 is a human immunodeficiency virus (HIV) entry inhibitor that is being developed for the oral treatment of HIV infection. Human plasma and urine LC/MS/ MS methods have been developed and validated for the quantitation of BMS-378806. For human plasma method, methyl t-butyl ether was used to extract BMS-378806 from plasma in a 96-well format, and the organic layers were dried down and then reconstituted for the injection, while a dilute-and-shoot approach was used for human urine method in a 96-well format. Chromatographic separation was achieved isocratically on a Phenomenex C18 (2) Luna column (2 x 50 mm2, 5 microm). The mobile phase contained 60:40 v/v of 0.1% formic acid in water and ACN. Detection was by positive ion electrospray MS/MS. The standard curves ranged from 1.25 to 1000 ng/mL for the plasma assay and from 10 to 5000 ng/mL for the urine assay. The curves were fitted to a 1/x2 weighted quadratic regression model for both methods. The validation results demonstrated that both methods had satisfactory precision and accuracy across the calibration ranges. The methods were applied to the analysis of human plasma and urine samples from a single ascending dose clinical study to assess the pharmacokinetics of the drug. The pharmacokinetic analysis results indicated the absorption and disposition of the drug was rapid. The systemic exposure of BMS-378806 was generally dose proportional among the doses from 100 to 1200 mg, but not dose proportional to 1600 mg. There were modest increases in the systemic exposure when the drug was given with food or given as a solution formulation. Renal excretion was not a substantial elimination pathway of the drug. BMS378806 was safe and well tolerated over a dose range of 100-1600 mg administered as a single oral dose.     

Sensitive and rapid method to determine trimetazidine in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive, and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method, using electrospray ionization, was developed and validated to quantify trimetazidine in human plasma using propranolol hydrochloride as an internal standard (IS). Samples were prepared by solid-phase extraction and analyzed without drying and reconstitution. The analyte and IS were chromatographed on a C18 reversed-phase column under isocratic conditions using 2 mM ammonium acetate (pH 3.5)-acetonitrile (40 + 60, v/v) as the mobile phase with a run time of 2.0 min. Quantitation was done on a triple-quadrupole mass analyzer API-3000, equipped with turbo ion spray interface and operating in multiple reaction monitoring mode to detect parent --> product ion (m/z 267.2 --> 181.4) transition. The method was validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, and stability. Linearity in plasma was observed over the concentration range of 1.5-300 ng/mL. Lower limit of quantification achieved was 1.5 ng/mL with precision < 10% using 10 microL injection volume. The mean relative recovery of analyte (97.36%) and IS (99.93%) was consistent and reproducible. Interbatch and intrabatch precision was < 8.0% and the accuracy determined was within +/- 8% in terms of relative error.     

Sensitive determination of free and plasma protein-bound dipyridamole by high-performance liquid chromatography

For many years dipyridamole (DP) has been used in the treatment of hypertension as a vasodilator, but recently it has been recognised as an anti-platelet aggregation agent and to potentiate anti-metabolite activity. A rapid and sensitive (20 nM) procedure for the determination of free and protein-bound DP in plasma, using reversed-phase high-performance liquid chromatography on an Ultrasphere XL ODS (3 microns) column (70 mm x 4.6 mm I.D.) with ultraviolet detection (280 nm), is reported. Free and bound DP were separated using ultrafiltration. Concentrations of DP between 0.1 and 10 microM were measured in plasma with a relative standard deviation of less than 9.6%. The subsequent determination of DP levels in patients orally administered 450 mg per day showed that DP binding to plasma protein is higher than 90%.     

A rapid and sensitive liquid chromatography/tandem mass spectrometry method for determination of docetaxel in human plasma

A novel, rapid and sensitive isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for quantification of docetaxel in human plasma with paclitaxel as internal standard. The high sensitivity and specificity of MS/MS detection enabled the use of a small volume of plasma (0.05 mL) and a simple liquid-liquid extraction procedure. Furthermore, a very short run-time (3 min) fulfilled the need for monitoring plasma levels of docetaxel from large-scale clinical studies. The calibration curve for docetaxel was linear over the range 5-1000 ng/mL with coefficients of correlation >0.999 using only 0.05 mL plasma. The intra- and inter-day precisions (CV) of analysis were <7%, and accuracy ranged from 96 to 110%. The applicability of the method was demonstrated in a pharmacokinetic study of a 1-h infusion of docetaxel with dosages of 75 mg/m(2). Possible conjugated metabolites of docetaxel were not detected in patients' samples.     

Monitoring stevioside in soju by high-performance liquid chromatography and liquid chromatography/mass spectrometry

A method using high-performance liquid chromatography (HPLC) with UV absorption detection was developed to monitor stevioside in soju, a distilled spirits product that is commercially available. The method uses a single-step dilution for sample preparation. It completely eliminates the time-consuming process of solid-phase extraction. A method using HPLC/mass spectrometry was optimized to confirm the identities of stevioside and other related impurities, including rebaudioside A, rebaudioside C, and dulcoside. The method was validated. The validation parameters included range (10.1-1007.3 ppm), precision, linearity, accuracy, robustness, system suitability, and intermediate precision. Stevioside standard solutions at 6 concentration levels were prepared for the validation work, including the tests for precision, linearity, and accuracy. The solutions were prepared in triplicate for each concentration. The relative standard deviation for the precision test was <3% for all 6 concentration levels. The correlation coefficient for the linearity within the concentration range was determined to be > 0.999. The average recovery ranged from 95.7 to 101.1% for the soju samples spiked with stevioside standard. The detection limit for stevioside was estimated at 75 ppb. The method was used to screen several soju samples; no detectable stevioside was found in the samples.     

High-throughput quantification for a drug mixture in rat plasma-a comparison of Ultra Performance liquid chromatography/tandem mass spectrometry with high-performance liquid chromatography/tandem mass spectrometry

A quantitative Ultra Performance liquid chromatography/tandem mass spectrometry (UPL/MS/MS) protocol was developed for a five-compound mixture in rat plasma. A similar high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) quantification protocol was developed for comparison purposes. Among the five test compounds, three preferred positive electrospray ionization (ESI) and two preferred negative ESI. As a result, both UPLC/MS/MS and HPLC/MS/MS analyses were performed by having the mass spectrometer collecting ESI multiple reaction monitoring (MRM) data in both positive and negative ion modes during a single injection. Peak widths for most standards were 4.8 s for the HPLC analysis and 2.4 s for the UPLC analysis. There were 17 to 20 data points obtained for each of the LC peaks. Compared with the HPLC/MS/MS method, the UPLC/MS/MS method offered 3-fold decrease in retention time, up to 10-fold increase in detected peak height, with 2-fold decrease in peak width. Limits of quantification (LOQs) for both HPLC and UPLC methods were evaluated. For UPLC/MS/MS analysis, a linear range up to four orders of magnitude was obtained with r2 values ranging from 0.991 to 0.998. The LOQs for the five analytes ranged from 0.08 to 9.85 ng/mL. Three levels of quality control (QC) samples were analyzed. For the UPLC/MS/MS protocol, the percent relative standard deviation (RSD%) for low QC (2 ng/mL) ranged from 3.42 to 8.67% (N = 18). The carryover of the UPLC/MS/MS protocol was negligible and the robustness of the UPLC/MS/MS system was evaluated with up to 963 QC injections.