Simple liquid chromatography method for the quantification of irinotecan and SN38 in sheep plasma: Application to in vivo pharmacokinetics after pulmonary artery chemoembolization using drug eluting beads

A rapid and simple liquid chromatography–fluorescence detection (LC–FD) method was developed and validated for the simultaneous quantification of irinotecan (CPT11) and SN38 in sheep plasma. Camptothecin (CPT) was used as the internal standard. A single step protein precipitation with acetonitrile was used for sample preparation. The separation was achieved using a 5 μm C18 column (250 mm × 4.5 mm, 5 μm) with a mobile phase composed of 36 mM sodium dihydrogen phosphate dehydrate and 4 mM sodium 1 heptane sulfonate–acetonitrile (72:28), the pH of the mobile phase was adjusted to 3. The flow rate was 1.45 mL/min and the fluorescence detection was operated at 355/515 nm (excitation/emission wavelengths). The run time was 13 min. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 5 ng/mL for both CPT11 and SN38. The assay was linear over concentrations ranging from 5 to 5000 ng/mL and to 240 ng/mL for CPT11 and SN38, respectively. This method was used successfully to perform plasma pharmacokinetic studies of CPT11 after pulmonary artery embolization (PACE) in a sheep model. It was also validated for CPT11 and SN38 analysis in sheep lymph and human plasma.     

Development of a sensitive and selective method for the quantitative analysis of cortisol,cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.     

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C₁₈ column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.     

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma

Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.     

Capillary zone electrophoresis with diode-array detection for analysis of local anaesthetics and opium alkaloids in urine samples

The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 μg mL^?1 in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ng mL^?1 for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Simultaneous determination of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma by liquid chromatography–mass spectrometry: Application to pharmacokinetics

A rapid high-performance liquid chromatography–mass spectrometry (HPLC–MS) method was developed and validated for simultaneous quantification of 6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol in rat plasma after oral administration of ginger oleoresin. Plasma samples extracted with a liquid–liquid extraction procedure were separated on an Agilent Zorbax StableBond-C₁₈ column (4.6 mm × 50 mm, 1.8 μm) and detected by MS with electrospray ionization interface in positive selective ion monitoring (SIM) mode. Calibration curves (1/x² weighted) offered satisfactory linearity (r² > 0.995) in a wide linear range (0.0104–13.0 μg/mL for 6-gingerol, 0.00357–4.46 μg/mL for 8-gingerol, 0.00920–11.5 μg/mL for 10-gingerol and 0.00738–9.22 μg/mL for 6-shogaol). The lower limit of quantification (LLOQ) was in a range of 3.57–10.4 ng/mL. The analytes and internal standard can be baseline separated within 6 min. Inter- and intra-day assay variation was less than 15%. This developed method was successfully applied to pharmacokinetic studies of ginger oleoresin after oral administration to rats. Glucuronide of 6-gingerol was determined after β-glucuronidase hydrolysis for more information, and the intestinal glucuronidation was further confirmed by comparison of plasma samples of hepatic portal vein and femoral vein.     

Rapid confirmatory method for the determination of sixteen synthetic growth promoters and bisphenol A in bovine milk using dispersive solid-phase extraction and liquid chromatography–tandem mass spectrometry

A rapid liquid chromatographic–tandem mass spectrometric (LC–MS/MS) multi-residue method for the simultaneous quantitation and identification of sixteen synthetic growth promoters and bisphenol A in bovine milk has been developed and validated. Sample preparation was straightforward, efficient and economically advantageous. Milk was extracted with acetonitrile followed by phase separation with NaCl. After centrifugation, the extract was purified by dispersive solid-phase extraction with C18 sorbent material. The compounds were analysed by reversed-phase LC–MS/MS using both positive and negative ionization and operated in multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for unambiguous confirmation. Total chromatographic run time was less than 10 min for each sample. The method was validated at a level of 1 μg L^?1. A wide variety of deuterated internal standards were used to improve method performance. The accuracy and precision of the method were satisfactory for all analytes. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC–MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be below the chosen validation level of 1 μg L^?1 for all compounds.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.     

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography–ion trap-tandem mass spectrometry coupled with electro spray ionization (LC–ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C₁₈ ACE^® column (50.0 mm × 2.1 mm, 5 μm, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3 mL/min. The transition monitored (positive mode) was m/z 279.1 → m/z 208.1 for triprolidine, m/z 165.9 → m/z 148.0 for pseudoephedrine and m/z 172.0 → m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0 min and a linear calibration curves ranged from 0.2 to 20.0 ng/mL for triprolidine and 5.0–500.0 ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3–106.3% and 1.0–9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.     

Simultaneous HPLC–UV analysis of rufinamide,zonisamide, lamotrigine,oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 μL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 μm Hydro-RP, 250 mm × 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2–40 μg/mL for RFN and 2–80 μg/mL for ZNS. The limit of quantitation was 2 μg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).     

Stability evaluation and sensitive determination of antiviral drug,valacyclovir and its metabolite acyclovir in human plasma by a rapid liquid chromatography–tandem mass spectrometry method

A simple, sensitive and high throughput liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC–ESI-MS/MS) method has been developed for the simultaneous determination of valacyclovir and acyclovir in human plasma using fluconazole as internal standard (IS). The method involved solid phase extraction of the analytes and IS from 0.5 mL human plasma with no reconstitution and drying steps (direct injection of eluate). The chromatographic separation was achieved on a Gemini C18 analytical column using isocratic mobile phase, consisting of 0.1% formic acid and methanol (30:70 v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for valacyclovir (m/z 325.2 → 152.2), acyclovir (m/z 226.2 → 152.2) and IS (m/z 307.1 → 220.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.0–1075 ng/mL and 47.6–10225 ng/mL for valacyclovir and acyclovir respectively. The mean recovery of valacyclovir (92.2%), acyclovir (84.2%) and IS (103.7%) from spiked plasma samples was consistent and reproducible. The bench top stability of valacyclovir and acyclovir was extensively evaluated in buffered and unbuffered plasma. It was successfully applied to a bioequivalence study in 41 healthy human subjects after oral administration of 1000 mg valacyclovir tablet formulation under fasting condition.     

Liquid chromatography and tandem mass spectrometry for the quantitative determination of ixabepilone (BMS-247550, Ixempra™) in human plasma: Method validation,overcoming curve splitting issues and eliminating chromatographic interferences from degradants

A sensitive method was developed and validated for the measurement of ixabepilone (BMS-247550, Ixempra?) using a demethylated analogue of ixabepilone (BMS-212188) as an internal standard. A 0.050 mL portion of each plasma sample was extracted with 0.450 mL of acetonitrile containing the internal standard via protein precipitation. The supernatant was analyzed on a LC–MS/MS system. Chromatography was carried out on a 2.0 mm × 100 mm YMC ODS-AQ 3 μm column using an isocractic mobile phase consisting of acetonitrile:10 mM ammonium acetate, pH 5.0 (70:30, v/v) at a flow rate of 0.30 mL/min. The mass spectrometer was fitted with a TurboIonSpray^® source and operated in negative ionization mode. Detection of ixabepilone and BMS-212188 were accomplished using multiple reaction monitoring (MRM) of precursor > product ion pairs of m/z 505.2 > 405.2, and 492.1 > 392.1, respectively. The assay range was 2.00–500 ng/mL and was fitted to a 1/x² weighted quadratic regression model. Replicate sample analysis indicated that intra- and inter-day accuracy and precision are within ±15.0%. The recovery of ixabepilone from 0.050 mL of plasma containing 5.00 and 400 ng/mL was greater than 94%. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies. This paper also discussed approaches used for resolving a curve splitting issue observed during quantitative analysis of ixabepilone in biological matrices. Finally, to adapt the methodology to pharmacokinetics of ixabepilone after oral administration, the potential interference of chemical degradants on the determination of ixabepilone was evaluated.     

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C₁₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00–1000 ng/mL and 5.00–5000 ng/mL for OLM in human plasma and urine as well as 0.500–200 ng/mL and 25.0–25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

Simultaneous determination of active xanthone glycosides,timosaponins and alkaloids in rat plasma after oral administration of Zi-Shen Pill extract for the pharmacokinetic study by liquid chromatography–tandem mass spectrometry

A sensitive and reliable liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) has been developed and validated for simultaneous determination of active components, i.e., xanthone glycosides (neomangiferin and mangiferin), timosaponins (timosaponin E1, timosaponin B-II and timosaponin B) and alkaloids (palmatine and berberine) in rat plasma after oral administration of Zi-Shen Pill extract. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards ginsenoside Re (for xanthone glycosides and timosaponins) and tetrahydroberberine (for alkaloids). LC separation was achieved on a Zorbax SB-C₁₈ column (150 mm × 2.1 mm I.D., 3.5 μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.25 mL/min. The detection was carried out by a triple–quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between negative (for xanthone glycosides and timosaponins) and positive (for alkaloids) ionization mode. Linear calibration curves were obtained over the concentration range of 5–1000 ng/mL for mangiferin, 0.5–100 ng/mL for neomangiferin, timosaponin E1, timosaponin B-II and timosaponin B, and 0.05–10 ng/mL for palmatine and berberine. The mean recovery of all the analytes ranged from 64.7 to 93.8%. The intra- and inter-day precision (% R.S.D.) was within 11.7% and accuracy (% bias) ranged from ?9.0 to 10.9%. This fully validated method was successfully applied to pharmacokinetic study of the above seven compounds in rats.     

A novel validated procedure for the determination of nicotine,eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry

A novel validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure was developed and fully validated for the simultaneous determination of nicotine-N-β-d-glucuronide, cotinine-N-oxide, trans-3-hydroxycotinine, norcotinine, trans-nicotine-1′-oxide, cotinine, nornicotine, nicotine, anatabine, anabasine and cotinine-N-β-d-glucuronide in human plasma or urine. Target analytes and corresponding deuterated internal standards were extracted by solid-phase extraction and analyzed by LC–MS/MS with electrospray ionization (ESI) using multiple reaction monitoring (MRM) data acquisition. Calibration curves were linear over the selected concentration ranges for each analyte, with calculated coefficients of determination (R²) of greater than 0.99. The total extraction recovery (%) was concentration dependent and ranged between 52–88% in plasma and 51–118% in urine. The limits of quantification for all analytes in plasma and urine were 1.0 ng/mL and 2.5 ng/mL, respectively, with the exception of cotinine-N-β-d-glucuronide, which was 50 ng/mL. Intra-day and inter-day imprecision were ≤14% and ≤17%, respectively. Matrix effect (%) was sufficiently minimized to ≤19% for both matrices using the described sample preparation and extraction methods. The target analytes were stable in both matrices for at least 3 freeze–thaw cycles, 24 h at room temperature, 24 h in the refrigerator (4 °C) and 1 week in the freezer (?20 °C). Reconstituted plasma and urine extracts were stable for at least 72 h storage in the liquid chromatography autosampler at 4 °C. The plasma procedure has been successfully applied in the quantitative determination of selected analytes in samples collected from nicotine-abstinent human participants as part of a pharmacokinetic study investigating biomarkers of nicotine use in plasma following controlled low dose (7 mg) transdermal nicotine delivery. Nicotine, cotinine, trans-3-hydroxycotinine and trans-nicotine-1′-oxide were detected in the particular sample presented herein. The urine procedure has been used to facilitate the monitoring of unauthorized tobacco use by clinical study participants at the time of physical examination (before enrollment) and on the pharmacokinetic study day.     

Determination of Tenacissoside A in rat plasma by liquid chromatography–tandem mass spectrometry method and its application to pharmacokinetic study

A sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for the first time for the estimation of Tenacissoside A in the rats’ plasma, which is the major active constituent in Marsdenia tenacissima. Tenacissoside A was extracted from the rats’ plasma by using liquid–liquid extraction (LLE), medroxyprogesterone acetate was used as the internal standard. An Alltech C18 column (250 mm × 4.6 mm, 5 μm) was used to provide chromatographic separation by detection with mass spectrometry operating in selected ion monitoring (SIM) mode. The method was validated over the concentration range of 1–250 ng/mL for Tenacissoside A. The precisions within and between-batch (CV%) were both less than 15% and accuracy ranged from 90 to 102%. The lower limit of quantification was 1 ng/mL and extraction recovery was 88.3% on average. The validated method was used to study the pharmacokinetic profile of Tenacissoside A in rat after administration.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.