Liquid chromatography–tandem mass spectrometry for the determination of paclitaxel in rat plasma after intravenous administration of poly(l-glutamic acid)-alanine-paclitaxel conjugate

A specific and sensitive liquid chromatography–tandem mass spectrometric method for quantitative determination of paclitaxel in rat plasma was developed and validated using docetaxel as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma. The separation of paclitaxel was performed on a C₁₈ column with a mobile phase of acetonitrile:water:formic acid (65:35:0.1, v/v/v) over 5 min. The assay was based on the selected reaction monitoring transitions at m/z of the precursor-product ion transitions m/z 854.2 → 286.1 for paclitaxel and 808.3 → 527.2 for internal standard. The lower limit of quantification was 0.5 ng/mL based on 100 μL of plasma. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 95.4 and 105.4%. The extraction recoveries ranged from 96.7 to 103.7% across the calibration curve range. The method was successfully applied to measurement of low concentrations of paclitaxel or regenerated paclitaxel in plasma after intravenous administration of a single dose (10 mg/kg) of a poly(l-glutamic acid)-alanine-paclitaxel conjugate to rats.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Development and validation of a LC–MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for the identification and quantification of bivalirudin in human plasma using nafarelin as the internal standard. Following protein precipitation with methanol, the analytes were separated on a C₁₈ column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Quantification of bivalirudin was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 1091.4 → (356.4 + 227.4) for bivalirudin and m/z 662.4 → 328.5 for IS. The lower limit of quantification was 1.25 ng/ml, and the assay exhibited a linear range of 1.25–500 ng/ml. The developed assay method was successfully applied to a pharmacokinetic (PK) study in healthy volunteers after intravenous administration of bivalirudin.     

Determination of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry

A sensitive and rapid method was developed for quantification of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS). An aliquot of 1 mL plasma sample was extracted with ethyl acetate–dichloromethane. Separation of olprinone and the milrinone (internal standard, IS) from the interferences was achieved on a C₁₈ column followed by MS/MS detection. The analytes were monitored in the positive ionization mode. Multiple reaction monitoring using the transition of m/z 251 → m/z 155 and m/z 212 → m/z 140 was performed to quantify olprinone and IS, respectively. The method had a total chromatographic run time of 3 min and linear calibration curves over the concentration range of 0.5–60 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-day precisions were less than 16.3% for low QC level, and 7.1% for other QC levels, respectively. The intra- and inter-day relative errors were ranged between −12.2% and 3.7% for three QC concentration levels. The validated method was successfully applied to the quantification of olprinone concentration in human plasma after intravenous (i.v.) administration of olprinone at a constant rate of infusion of 2 μg/(kg min) for 5 min in order to evaluate the pharmacokinetics.     

Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry

Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC–MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C₁₈ column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R = 0.9967) for plasma samples and 10 to 6000 ng/mL (R ≥ 0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5–104.6% and 93.3–107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T_max, 10.7 ± 0.31 min; t_1/2, 51.40 ± 6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.     

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C₁₈ column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10-1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.     

Liquid chromatograph/tandem mass spectrometry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study

A rapid and high sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous determination of chlorogenic acid and cinnamic acid in human plasma was developed. The analytes and internal standard (IS), tinidazole, were extracted from human plasma via liquid/liquid extraction with ether–ethyl acetate (1:1, v/v) and separated on an Agilent Zorbax SB C18 column within 5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM) and negative ion mode. The precursor to product ion transitions monitored for chlorogenic acid, cinnamic acid and IS were m/z 352.9 → 191.1, 146.8 → 103.1, 245.6 → 126.0, respectively. The assay was validated with linear range of 1.00–800.00 ng/mL for chlorogenic acid and 0.50–400.00 ng/mL for cinnamic acid. The intra- and inter-day precisions (RSD%) were within 9.05% for each analyte. The absolution recoveries were greater than 74.62% for chlorogenic acid and 76.21% for cinnamic acid. Each analyte was proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study of Mailuoning injection in 10 healthy volunteers.     

Quantitative analysis of colistin A and colistin B in plasma and culture medium using a simple precipitation step followed by LC/MS/MS

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm × 250 mm, 5 μm particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor–product ion pairs were m/z 1167.7 → 1079.6 for colistin A and m/z 1153.7 → 1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 μL plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV <6.2% and accuracy < ±12.6%. For culture medium (50 μL + 50 μL plasma), LLOQ was 24.2 and 13.2 ng/mL for colistin A and B, respectively, with CV <11.4% and accuracy < ±8.1%. The quick sample work-up method allows for determination of colistin A and B in clinical samples without causing hydrolysis of the prodrug colistin methanesulfonate (CMS).     

Validated LC–MS/MS method for the determination of sarpogrelate in human plasma: Application to a pharmacokinetic and bioequivalence study in Chinese volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the determination of sarpogrelate in human plasma. One-step protein precipitation with acetonitrile was used to extract the analytes from the plasma. Sarpogrelate and tramadol (internal standard, I.S.) were separated on a Venusil MP-C₁₈ column within 1.7 min, using acetonitrile:ammonium acetate (10 mM, pH 6.8) (55:45, v/v) as mobile phase at a flow rate of 1.2 mL/min with an approximately 1:1 split entering the mass spectrometer. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of sarpogrelate at m/z 430.3 → 135.3 and of I.S. at m/z 264.1 → 58.0. The assay was validated over the concentration range of 1–1000 ng/mL with a lower limit of quantitation (LLOQ) of 1 ng/mL using 50 μL of plasma. The intra- and inter-day precision (relative standard deviation, R.S.D.) were ≤6.4% and ≤5.4%, respectively, with accuracy (relative error, R.E.) in the range 0.5–3.6%. The method was successfully applied to a pharmacokinetic and bioequivalence study enrolling 22 Chinese volunteers administered sarpogrelate tablets.     

Quantification of cinacalcet by LC-MS/MS using liquid-liquid extraction from 50 μL of plasma

A simple and economical high-performance liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of cinacalcet in plasma. Following liquid-liquid extraction, the analyte was separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 358-155 for cinacalcet and m/z 310-148 for the internal standard. The assay exhibited a linear dynamic range of 0.1-200 ng/mL for cinacalcet in plasma. Acceptable precision (<10%) and accuracy (100 ± 5%) were obtained for concentrations over the standard curve range. A run time of 3.5 min for each sample made it possible to analyze more than 250 samples per day. The method was successfully applied to quantify cinacalcet concentrations in a preclinical pharmacokinetic study after a single oral administration of cinacalcet at 10 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C_max; 160 ± 56 ng/mL) was achieved at 1.0 h (T_max), area under the curve (AUC) and half-life (t_1/2) were 949 ± 257 ng h/mL and 3.58 ± 0.4 h, respectively.     

Rapid,sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of topically applied azithromycin in rabbit conjunctiva tissues

A simple, rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of azithromycin in rabbit conjunctiva tissues using roxithromycin as internal standard. Following a deproteinization procedure, the samples were eluted isocratically at a flow rate of 0.3 mL/min utilizing a mobile phase containing of 10 mM ammonium acetate (adjusted pH to 5.2 with 0.1% acetic acid)–methanol (18:82, v/v) and a SHISEIDO CAPCELL PAK C₁₈ (3.0 mm × 75 mm, 3 μm). Azithromycin and its internal standard were measured by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode with precursor-to-product qualifier transition m/z 375 [M+2H]²⁺ → 591 and m/z 837 [M+H]⁺ → 679 respectively. The method demonstrated that good linearity ranged from 10 to 2000 ng/mL with r = 0.9998. The lower limit of quantification for azithromycin in conjunctiva tissues was 10 ng/mL with good accuracy and precision. The intra- and inter-day precision (RSD) values were below 15% and accuracy (%) ranged from 90% to 110% at all QC levels. The method was applicable to ocular pharmacokinetic studies of azithromycin.     

Liquid chromatography–tandem mass spectrometry for the simultaneous quantitation of artemether and lumefantrine in human plasma: Application for a pharmacokinetic study

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the simultaneous quantitation of artemether and lumefantrine in human plasma was developed and validated. Artesunate was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure and separated on a reversed-phase Zorbax SB-Ciano column with a mobile phase composed of methanol and 10 mM aqueous ammonium acetate containing 0.2% (v/v) acetic acid and 0.1% (v/v) formic acid. Multiple reaction monitoring was performed using the transitions m/z 316 → m/z 267, m/z 530 → m/z 348 and m/z 402 → m/z 267 to quantify artemether, lumefantrine and artesunate, respectively. Calibration curves were constructed over the range of 10–1000 ng/mL for artemether and 10–18,000 ng/mL for lumefantrine. The lower limit of quantitation was 10 ng/mL for both drugs. The mean R.S.D. values for the intra-run precision were 2.6% and 3.0% and for the inter-run precision were 3.6% and 4.6% for artemether and lumefantrine, respectively. The mean accuracy values were 102.0% and 101.2% for artemether and lumefantrine, respectively. No matrix effect was detected in the samples. The validated method was successfully applied to determine the plasma concentrations of artemether and lumefantrine in healthy volunteers, in a one-dose pharmacokinetic study, over the course of 11 days.     

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.     

Determination of strychnine and brucine in rat plasma using liquid chromatography electrospray ionization mass spectrometry

A simple, sensitive and selective liquid chromatography–electrospray mass spectrometric (LC–ESI-MS) method was developed and validated for simultaneous determination of strychnine and brucine in rat plasma, using tacrine as the internal standard (IS). Sample preparation involved a liquid–liquid extraction of the analytes with n-hexane, dichloromethane and isopropanol (65:30:5, v/v/v) from 0.1 mL of plasma. Chromatographic separation was carried out on a Waters C₁₈ column using a mobile phase of methanol–20 mM ammonium formate–formic acid (32:68:0.68, v/v/v). Positive selected ion monitoring mode was used for detection of strychnine, brucine and the IS at m/z 335.2, m/z 395.2 and m/z 199.2, respectively. Linearity was obtained over the concentration range of 0.5–500 ng/mL for strychnine and 0.1–100 ng/mL for brucine. The lower limit of quantification was 0.5 ng/mL and 0.1 ng/mL for strychnine and brucine, respectively. The intra- and inter-day precision for both strychnine and brucine was less than 7.74%, and accuracy ranged from −4.38% to 2.21% at all QC levels. The method has been successfully applied to a pharmacokinetic study of processed Semen Strychni after oral administration to rats.     

Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma using HPLC coupled with tandem mass spectrometry: Application to bioequivalence studies

A sensitive, specific and selective liquid chromatography/tandem mass spectrometric method has been developed and validated for the simultaneous determination of irbesartan and hydrochlorothiazide in human plasma. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard losartan were separated on a reverse phase C₁₈ column (50 mm × 4 mm, 3 μm) using water with 2.5% formic acid, methanol and acetonitrile (40:45:15, v/v/v (%)) as a mobile phase (flow rate of 0.70 mL/min). Irbesartan and hydrochlorothiazide were ionized using ESI source in negative ion mode, prior to detection by multiple reaction monitoring (MRM) mode while monitoring at the following transitions: m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide, 427 → 175 for irbesartan. Linearity was demonstrated over the concentration range 0.06–6.00 μg/mL for irbesartan and 1.00–112.00 ng/mL for hydrochlorothiazide. The developed and validated method was successfully applied to a bioequivalence study of irbesartan (300 mg) with hydrochlorothiazide (12.5 mg) tablet in healthy volunteers (N = 36).     

Oltipraz promotion of liver regeneration after partial hepatectomy: The role of PI3-kinase-dependent C/EBPβ and cyclin E regulation

Oltipraz, a representative cancer chemopreventive agent, regenerates cirrhotic liver via CCAAT/enhancer binding protein β (C/EBPβ). This study examined the effect of oltipraz on liver regeneration after partial hepatectomy (PH) and explored the role of phosphatidylinositol 3-kinase (PI3K) pathway responsible in liver regeneration. Oltipraz treatment (30 mg/kg/day, for 3 days) promoted liver regeneration in PH rats, but did not increase hepatocyte growth factor production. Subcellular fractionation and electrophoretic mobility shift assays showed that oltipraz treatment increased C/EBPβ-DNA binding activity in the liver of sham control rats and further enhanced PH-mediated nuclear translocation of C/EBPβ. The expression of cyclin E and the activity of cyclin E-dependent kinase were both enhanced by oltipraz treatment of PH rats. The signaling pathway that controls C/EBPβ and cyclin E were studied in H4IIE cells, a rat-derived hepatocyte cell line. Oltipraz potentiated the nuclear accumulation of C/ EBPβ and C/EBPβ-DNA binding activity in cells incubated in a medium containing serum. PI3K and its downstream kinase, p70S6 kinase, were both required for C/EBPβ-dependent induction of cyclin E by oltipraz, as shown by chemical inhibition and plasmid transfection experiments. The results of this study demonstrate that oltipraz treatment enhances liver regeneration after PH, which involves activation of C/EBPβ and C/EBPβ-dependent cyclin E expression via the PI3K-p70S6 kinase pathway.     

Simultaneous quantification of losartan and active metabolite in human plasma by liquid chromatography–tandem mass spectrometry using irbesartan as internal standard

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method employing electronspray ionization was developed and validated for quantification of losartan and its carboxylic acid metabolite in human plasma using irbesartan as internal standard (IS). Following a simple pretreatment procedure, the analytes were separated using a gradient mobile phase on reverse phase C₁₈ column. Selected reaction monitoring was specific for losartan, losartan acid and irbesartan. The method validation demonstrated the specificity, lower limit of quantification, accuracy and precision of measurements. The assay exhibited a linear dynamic range of 2.0–400 ng/mL for losartan and 1.85–370 ng/mL for losartan acid. A run time of 3.5 min for each sample made it possible to analyze more than 200 samples per day. The validated method has been successfully used to analyze human plasma samples for application in bioavailability/bioequivalence studies.     

Determination of oxymatrine and its metabolite matrine in rat blood and dermal microdialysates by high throughput liquid chromatography/tandem mass spectrometry

Oxymatrine (OMT) and matrine (MT) are the major quinolizidine alkaloids found in certain Sophora plants, which have been extensively used in China for the treatment of viral hepatitis, cancer, cardiac diseases and skin diseases (such as atopic dermatitis and eczema). A precise, sensitive and high throughput LC–MS/MS was developed to determine OMT and its metabolite MT in rat blood and dermis collected using microdialysis technique. Microdialysis probes were inserted into the jugular vein/right atrium and dermis of Wistar rats, and 3% OMT gel (1 g) was administered via topical application. The samples were collected and then injected into the LC–MS/MS system after adding the internal standard (codeine, CDN). Chromatographic separation was achieved in a run time of 2 min on a reversed phase short-column (50 mm × 2.1 mm, 3.5 μm). The mobile phase for column separation was methanol–ammonium formate (pH 5.0; 25 mM) (70:30, v/v) with a flow rate of 0.3 mL/min. A diverter valve was installed post-LC column for desalting. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for OMT, MT and IS was m/z 265.0 → 247.3, 249.1 → 148.3 and 300.0 → 215.2, respectively. The lower limit of quantification (LLOQ) for OMT and MT was 0.5 ng/mL. The calibration curves were linear over the range of 0.5–1000 ng/mL for OMT and MT with a coefficient of determination >0.999. This selective and sensitive method is useful for the determination of OMT and MT and in the pharmacokinetic studies of these compounds. The blood and dermal concentration–time profile of OMT and its metabolite MT suggest that the limiting factor for dermal metabolism is the low capacity of enzymes in the skin rather than the quantity of penetrated OMT.     

Quantitative determination of pimozide in human plasma by liquid chromatography–mass spectrometry and its application in a bioequivalence study

A simple, sensitive and specific LC–ESI/MS method was developed for the determination of pimozide in human plasma. Pimozide and cinnarizine (internal standard) were isolated from plasma samples by liquid–liquid extraction. The chromatographic separation was accomplished on a Thermo Hypersil-HyPURITY C18 reversed-phase column (150 mm × 2.1 mm, i.d., 5 μm) with the mobile phase consisting of 5 mM ammonium acetate (pH 3.5, adjusted with acetic acid)–methanol–acetonitrile (39:5:56, v/v/v). The lower limit of quantification was 0.02 ng/mL, and the assay exhibited a linear range of 0.025–12.800 ng/mL. The established method has been successfully applied to a bioequivalence study of 2 pimozide formulations in 32 healthy male Chinese volunteers.     

Simultaneous quantitation of levodopa and 3-O-methyldopa in human plasma by HPLC-ESI-MS/MS: application for a pharmacokinetic study with a levodopa/benserazide formulation

A sensitive and simple method was developed for the quantitation of levodopa and its metabolite 3-O-methyldopa, in human plasma, after oral administration of tablet formulations containing levodopa (200 mg) and benserazide (50 mg). The analytes were extracted by a protein precipitation procedure, using carbidopa as an internal standard. A mobile phase consisting of 0.2% formic acid and acetonitrile (94:6, v/v) was used and chromatographic separation was achieved using ACE C₁₈ column (50 mm × 4.6 mm i.d.; 5 μm particle size). Selected reaction monitoring was performed using the fragmentation transitions m/z 198 → m/z 107, m/z 212 → m/z 166 and m/z 227 → m/z 181 for levodopa, 3-O-methyldopa and carbidopa, respectively. Calibration curves were constructed over the range 50.0-6000.0 ng/mL for levodopa and 25.0-4000.0 ng/mL for 3-O-methyldopa. The method shown to be specific, precise, accurate and provided recovery rates higher than 85% for all analytes. No matrix effect was detected in the samples. The validated method was applied in a pharmacokinetic study with a levodopa/benserazide tablet formulation in healthy volunteers.