LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

Simultaneous Determination of Pethidine and Atropine in Rabbit Plasma by LC�CMS�CMS and Its Application to a Pharmacokinetic Study after Intramuscular Administration

A sensitive and selective liquid chromatography?Ctandem mass spectrometry method for the determination of pethidine and atropine in rabbit plasma was developed and validated. The analytes and internal standard (IS) are extracted from plasma by liquid?Cliquid extraction using ethyl acetate, and separated on a Zorbax SB-Aq column (2.1 × 150 mm, 3.5 ??m) using acetonitrile?C0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode, and multiple reaction monitoring mode was used for quantification using target fragment ions m/z 247.8 ?? 219.7 for pethidine, m/z 289.9 ?? 123.8 for atropine and m/z 295.0 ?? 266.8 for IS, respectively. The assay is linear over the range of 5?C1,000 ng mL^?1 for pethidine and atropine, with a lower limit of quantification of 3 ng mL^?1 for pethidine and 5 ng mL^?1 for atropine. Intra-day and inter-day precision are less than 11% and the accuracy are in the range of 90.4?C106.3%. Furthermore, the newly developed method is successfully used for the determination of pethidine and atropine in rabbit plasma for pharmacokinetic study.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

LC–MS–MS Determination of Troxerutin in Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the determination of troxerutin in human plasma using tramadol as internal standard (IS) has been developed and validated. Sample preparation involved liquid–liquid extraction with ethyl acetate–isopropanol (95:5, v/v). The analyte and IS were separated by RP–LC with gradient elution using 10 mM ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.9 mL min^?1. LC–MS–MS in the positive ion mode employed multiple reaction monitoring of the transitions at m/z 743.2→435.3 and m/z 264.1→58.0 for troxerutin and IS, respectively. The assay was linear in the concentration range 0.01–10 ng mL^?1 with precision and accuracy within assay variability limits as per FDA guidelines. The assay was successfully applied to a pharmacokinetic study involving oral administration of 300 mg troxerutin to eight healthy Chinese volunteers.     

Sensitive and Selective LC-MS-MS Assay for the Quantification of Palonosetron in Human Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive and selective liquid chromatography-tandem mass spectrometry method was developed for the determination of palonosetron in human plasma samples. Chromatographic conditions and mass spectral parameters were optimized in order to achieve a limit of quantification of approximately 0.03 ng mL^?1. Palonosetron and citalopram (internal standard) were extracted by liquid–liquid extraction under alkaline conditions using saturated sodium bicarbonate. Separation was achieved with a Hanbon Lichrospher C₁₈ column and detection was carried out by tandem mass spectrometry using positive electrospray ionization in selected reaction monitoring mode. The target ions of palonosetron and citalopram were to m/z 297.00 → 297.00 and 325.00 → 325.00 respectively. Calibration curves were linear over the range of approximately 0.03–10 ng mL^?1. Precision and accuracy of this method was acceptable. The method was successfully applied to a pharmacokinetic study with healthy Chinese volunteers after intravenous administration of a single dose of 0.125, 0.25 or 0.5 mg palonosetron hydrochloride.     

LC�CMS�CMS Method for Quantification of Hydralazine in BALB/C Mouse Plasma and Brain: Application to Pharmacokinetic Study

A rapid, sensitive and accurate high performance liquid chromatography method using tandem mass spectrometry detection for hydralazine in BALB/C mouse plasma and brain was developed and validated. The method involved a derivatization with 2,4-pentanedione at 50 °C for 1 h, and a step of solid phase extraction to purify and concentrate hydralazine derivative. Chromatographic separation was carried out on an Agilent ZORBAX SB-C18 column by elution with methanol?C0.01 mol L^?1 ammonium acetate (60:40, v/v). The multiple reaction monitoring transition used for quantification was m/z 225.2 ?? 129.5 in the electrospray positive ionization mode. Good linearity was obtained over the concentration range of 10?C200 ng mL^?1. The limits of detection were 0.49 and 1.05 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. The limits of quantitation were 1.5 and 3.18 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. Sample analysis time was 6 min including sample separation. The method was successfully applied to a pharmacokinetic study following intraperitoneal injection of hydralazine in BALB/C mice at the dose of 20 mg kg^?1.     

Simultaneous Quantification of Oroxylin A and Its Metabolite Oroxylin A-7-O-Glucuronide: Application to a Pharmacokinetic Study in Rat

A sensitive liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed and validated for the quantification of cepharanthine (CEP) in beagle dog plasma. The chromatographic separation was performed on an Agilent-C18 column and the mobile phase was composed of methanol:water with 10 mM ammonium acetate (20:80, v/v). Detection was operated in the positive ion mode and the tandem mass spectrometer was tuned in the multiple reactions monitoring mode (MRM) to monitor m/z transitions 607 ?? 365 for CEP and 285 ?? 193 for the internal standard (IS) diazepam. This method exhibited a linear range of 5?C2,500 ng mL^?1. The precision (RSD%) and accuracy (RME%) of the assay were <8.7 and 2.4%, respectively. The limit of quantification was 5 ng mL^?1 and no significant matrix effect was observed. The validated method has been successfully applied to pharmacokinetic study of CEP in beagle dog.     

Validated Liquid–Liquid Extraction and LC–ESI–MS Method for the Determination of Melitracen in Human Plasma

Melitracen and the internal standard (I.S.), trifluoperazine, were extracted from plasma by a convenient liquid-liquid extraction. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C₁₈ with the mobile phase consisting of 10 mM ammonium acetate–methanol–acetonitrile. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M + H]⁺ ions at 292 m/z for melitracen and 408 m/z for trifluoperazine. The method was validated over 0.4–50.0 ng mL^?1 for melitracen. The recovery was 73.52–78.91%, and the lower limit of quantitation (LLOQ) detection was 0.4 ng mL^?1 for melitracen. The intra- and inter-day precision of the method at three concentrations were 2.96–7.76% with accuracy of 95.75–100.48%. Stability of compounds was established in a battery of stability studies. The bioequivalence of melitracen in the two formulations was evaluated in 18 healthy Chinese male volunteers with this assay. The described method showed acceptable precision, accuracy, linearity, stability, specificity and can be widely used for pharmacokinetic studies, and routine therapeutic drug monitoring.     

LC–MS Determination and Pharmacokinetic Study of Salidroside in Rat Plasma after Oral Administration of Traditional Chinese Medicinal Preparation Rhodiola crenulata Extract

A simple, rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed for the quantification of salidroside in rat plasma and the study of its pharmacokinetics after oral administration of 15 g kg^?1 Rhodiola crenulata extract to Wistar rats. A 200 μL plasma sample was extracted by acetonitrile and performed on Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with the mobile phase of acetonitrile–water (11:89) within a run time of 8 min. The analyte was monitored with electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 299.20 for salidroside and m/z 150.00 for internal standard (IS) paracetamol. A good linear relationship was obtained over the range of 100–20,000 ng mL^?1 and the lower limit of quantification was 100 ng mL^?1. The validated method was successfully applied for the pharmacokinetic study of salidroside in rat. After oral administration of Rhodiola crenulata extract, the main pharmacokinetic parameters T _max, T _1/2, C _max, AUC _0?t and AUC _0?∞ were 0.56 ± 0.21 h, 7.91 ± 4.42 h, 3,386 ± 2,138 ng mL^?1, 16,146 ± 6,558 ng h mL^?1 and 18,599 ± 6,529 ng h mL^?1, respectively.     

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

Highly Sensitive Analysis of Norethisterone in Human Serum by LC Coupled with Atmospheric Pressure Photoionization Tandem Mass Spectrometry

High-performance liquid chromatography coupled with tandem mass spectrometry has been used for sensitive and specific quantitative analysis of norethisterone (NE) in human serum. NE and the internal standard fentanil were isolated by solid-phase extraction. Chromatographic separation was achieved on a 4.6 mm × 50 mm, 5-μm particle, C₁₈ column. The mobile phase was 70:30 (v/v) methanol–0.5% aqueous formic acid. NE and the internal standard were detected by multiple-reaction monitoring of precursor/product ion combinations at m/z 299.4/231.2 and 377.1/188.1, respectively; an atmospheric-pressure-photoionization source was used in positive-ion mode. Linearity was established in the concentration range 0.2–49.24 ng mL^?1 and the lowest limit of quantification was 0.2 ng mL^?1. Recovery of NE ranged from 92.54 to 102.74% and relative standard deviations were <15%. The method was used for a pharmacokinetic study of NE in healthy postmenopausal Chinese female volunteers.     

LC–MS Simultaneous Determination of Itopride Hydrochloride and Domperidone in Human Plasma

A rapid, simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous quantification of itopride hydrochloride and domperidone in human plasma. Both drugs were extracted by liquid–liquid extraction with ethyl acetate and saturated borax solution. The chromatographic separation was performed on a reversed-phase C18 column with a mobile phase of water–methanol (2:98, v/v) containing 0.5% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The assay exhibited linearity over the concentration range of 3.33–500 ng mL^?1 for itopride hydrochloride and 3.33–100 ng mL^?1 for domperidone in human plasma. The precursor to product ion transitions of m/z 359.1–72.3 and 426.0–147.2 were used to measure itopride hydrochloride and domperidone respectively. The method was found suitable for the analysis of plasma samples collected during phase 1 pharmacokinetics study of itopride HCl 50 mg and domperidone 20 mg in 12 healthy volunteers after single oral doses of the combination drug.     

LC�CMS Analysis of Sertraline and Its Active Metabolite in Human Serum Using a Silica Column with a Non-Aqueous Polar Mobile Phase

A sensitive liquid chromatography?Cmass spectrometry method for the simultaneous determination of sertraline (SER) and its major metabolite norsertraline (NOR) from serum was developed and validated in the context of a pharmacokinetic study in pregnant women. The separations were achieved on a silica column with a non-aqueous polar mobile phase consisting of acetonitrile, methanol and ammonium acetate at a flow rate of 0.5 mL min^?1. The concentrations were measured using a single quadruple mass spectroscopic detector supplied with atmospheric pressure ionization electrospray. Sample preparation consisted of a simple liquid?Cliquid procedure. The detector was set in selective ion mode for each compound of interest, 306 m/z for SER and 275 m/z for NOR. Calibration curves were generated by least square linear regression for concentration of 5?C160 ng mL^?1 for SER and from 10 to 320 ng mL^?1 for NOR. The curves for both compounds of interest were linear, with correlation coefficients r ² ?? 0.999.     

Rapid and Selective LC�CMS�CMS Method for the Determination of cyclo-trans-4-l-Hydroxyprolyl-l-serine (JBP485) in Rat Plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of JBP485 was developed and validated. Following protein precipitation, the analyte and internal standard (JBP923) were separated from human plasma using an isocratic mobile phase on an Elite Kromasil C18 column. An API 3200 tandem mass spectrometer equipped with a Turbo ionSpray ionization source was used as the detector and operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 201.2 ?? 86.2 and m/z 219.2 ?? 86.2 was performed to quantify JBP485 and JBP923, respectively. The method was linear in the concentration range of 0.10?C50.00 ??g mL^?1 using 100 ??L of plasma. The lower limit of quantification was 0.10 ??g mL^?1. The intra- and inter-day relative standard deviations over the entire concentration range were less than 6.65%. Accuracy determined at three concentrations (0.25, 4.00 and 25.00 ??g mL^?1 for JBP485) ranged from ?0.78 to 2.74% in terms of relative error. Each plasma sample was chromatographed within 2.0 min. The method was successfully applied to characterize the pharmacokinetic profiles of JBP485 in rats after an intravenous injection of 6.25 mg kg^?1 JBP485.     

Determination of Memantine in Human Plasma by LC–MS–MS: Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of memantine was developed and validated over the linearity range 0.1–25 ng mL^?1 with 0.5 mL of plasma using procainamide as the internal standard. This analysis was carried out on a Cosmosil 5C₁₈-MS column and the mobile phase was composed of methanol: 0.5% formic acid (50:50, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization and quantification was performed by multiple reaction monitoring mode. The MS–MS ion transitions monitored were m/z 180 → 107 and 236 → 163 for memantine and procainamide, respectively. The between- and within-day precision was less than 10.9% and accuracy was less than 2.5%. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of memantine in healthy Chinese volunteers.     

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C₁₈ column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL^?1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL^?1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.     

Rapid and Sensitive LC−MS–MS Method for the Determination of Sarpogrelate in Human Plasma

A rapid and sensitive liquid chromatographic–tandem mass spectrometric method has been developed and validated for the estimation of sarpogrelate in human plasma. Sarpogrelate was extracted from human plasma by solid-phase extraction. Temocapril was used as the internal standard. Heated electron spray ionization mass spectrometry was performed on a TSQ Quantum Ultra MS system. The LC column was a Hypurity C₁₈ and the mobile phase was 2 mM ammonium formate (pH 3.00 ± 0.05):acetonitrile (30:70 v/v). A flow rate of 0.250 mL min^?1 was used. The quantitative analyses were carried out in the positive ion and full scan mode over the mass range m/z 60–500. The capillary, vaporiser temperatures were 325 and 200 °C respectively. The sheath gas pressure, spray voltage, collision energy and tube lense were 40, 3,500 V, 19 V, 198 V, respectively, and the mass spectra of the drugs were recorded by total ion monitoring. Retention times and characteristic mass fragments were recorded and the chosen diagnostic mass fragments were monitored in the mass chromatography mode. Signal intensities of each of the mass fragments: m/z 477 [M + H]⁺ for temocapril, m/z 430 [M + H]⁺ for sarpogrelate, were used for quantification. The calibration curves (the ratio between the peak areas as signal intensities of the drug analyzed and that of the internal standard (temocapril: m/z 477 [M + H]⁺) vs. the concentration of drug) exhibited linearity over the concentration range 5.00–2,500.00 ng mL^?1 human plasma. The recovery and the accuracy were calculated by comparing the peak areas as the signal intensities of each mass fragment for the drug in spiked samples after solid-phase extraction from human plasma to the peak area as the signal intensity of the mass fragment of internal standard sample. The method involves a rapid solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 5.0–2,500.0 ng mL^?1. The absolute recoveries for sarpogrelate (93.72%) and IS (91.42%) achieved from spiked plasma samples were consistent and reproducible.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

Simultaneous LC–MS–MS Analysis of Danshensu, Salvianolic Acid B, and Hydroxysafflor Yellow A in Beagle Dog Plasma, and Application of the Method to a Pharmacokinetic Study of Danhong Lyophilized Powder for Injection

A reliable and sensitive liquid chromatographic–tandem mass spectrometric method, with rutin as internal standard, has been developed and validated for simultaneous determination of danshensu, salvianolic acid B (SAB), and hydroxysafflor yellow A (HSYA) in beagle dog plasma. Plasma samples spiked with the analytes were extracted by solid-phase extraction and the analytes were separated on a 250 × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–acetonitrile–0.5% formic acid 20:25:55 (v/v) as mobile phase at a flow rate of 1 mL min^?1. LC–MS–MS analysis was performed with a Finnigan TSQ triple-quadrupole tandem mass spectrometer operated in negative-ion selected-reaction-monitoring mode, using electrospray ionization. The accuracy and precision of the method were acceptable and linearity was good over the range 20–4,000 ng mL^?1 for danshensu, 50–10,000 ng mL^?1 for SAB, and 10–2,000 ng mL^?1 for HSYA. The method was successfully applied to a pharmacokinetic study of a traditional Chinese medicinal preparation, Danhong lyophilized powder for injection.     

Quantification of Clarithromycin in Human Plasma by UPLC-MS-MS

A selective, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed for the quantitative determination and pharmacokinetic study of clarithromycin in human plasma. The analyte was extracted from human plasma under alkaline condition with diethyl ether. Separation was performed on an ACQUITY UPLC BEH C₁₈ column (50 mm × 2.1 mm i.d., 1.7 μm) with gradient elution at a flow-rate of 0.30 mL min^?1. The mobile phase was 50 mM ammonium acetate (pH 6.8) and acetonitrile. The detection was performed on a triple-quadruple tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization. Linear calibration curves were obtained in the concentration range of 1–3,000 ng mL^?1, with a lower limit of quantification (LLOQ) of 1 ng mL^?1. The intra- and inter-day precision (RSD) values were below 10% and accuracy (RE) ranged from ?7.2 to 6.1% at all QC levels. The method was utilized to support clinical pharmacokinetic study in healthy volunteers following oral administration of clarithromycin extended release tablets.