Determination of scopolamine in human serum and microdialysis samples by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC–MS–MS) method with a rapid and simple sample preparation was developed for the determination of scopolamine in biological fluids. Scopolamine and the internal standard atropine in serum samples were extracted and cleaned up by using an automated solid phase extraction method. Microdialysis samples were directly injected into the LC–MS system. The mass spectrometer was operated in the multi reaction monitoring mode. A good linear response over the range of 20 pg/ml to 5 ng/ml was demonstrated. The accuracy for added scopolamine ranged from 95.0 to 104.0%. The lower limit of quantification was 20 pg/ml. This method is suitable for pharmacokinetic studies.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Determination of bicyclol in dog plasma using liquid chromatography–tandem mass spectrometry

A sensitive and accurate method for determination of bicyclol in dog plasma was developed. Thermo Scientific TSQ Quantum triple quadrupole system with multiple ion monitoring (MIM) positive scanning mode was applied. Bicyclol and DDB (IS) sodium adduct molecular ions were monitored at m/z 413 and m/z 441 in both Q1 and Q3, respectively. The collision energy in Q2 was set to 15 eV. Precipitation method was employed in the extraction of bicyclol and DDB from the biological matrix. The method was validated over 1–500 ng/mL for bicyclol. The recovery was 96.5–109.5%, and the limit of quantitation (LOQ) detection was 1 ng/mL for bicyclol. The intra- and inter-day precision of the method at three concentrations was 3.3–14.3% with accuracy of 99.9–109.0%. The method was successfully applied to bioequivalence studies of bicyclol controlled-release formulation to obtain the pharmacokinetic parameters.     

Class-specific molecularly imprinted polymers for the selective extraction and determination of sulfonylurea herbicides in maize samples by high-performance liquid chromatography–tandem mass spectrometry

A novel method based on the molecularly imprinted solid-phase extraction (MISPE) procedure has been developed for the simultaneous determination of concentrations of sulfonylurea herbicides such as chlorsulfuron (CS), monosulfuron (MNS), and thifensulfuron methyl (TFM) in maize samples by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). The molecularly imprinted polymer (MIP) for sulfonylurea herbicides was synthesized by precipitation polymerization using chlorsulfuron as the template molecule, 2-(diethylamino)ethyl methacrylate (DEAMA) as the functional monomer, and trimethylolpropane trimethacrylate (TRIM) as the cross-linker. The selectivities of the chlorsulfuron template and its analogs on the molecularly imprinted polymer were evaluated by high-performance liquid chromatography (HPLC). The extraction and purification procedures for the solid-phase extraction (SPE) cartridge with a molecularly imprinted polymer as the adsorbent for the selected sulfonylurea herbicides were then established. A molecularly imprinted solid-phase extraction method followed by high-performance liquid chromatography–tandem mass spectrometry for the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl was also established. The mean recoveries of these compounds in maize were in the range 75–110% and the limits of detection (LOD) of chlorsulfuron, monosulfuron, and thifensulfuron methyl were 0.02, 0.75, and 1.45 μg kg⁻¹, respectively. It was demonstrated that the MISPE–HPLC–MS/MS method could be applied to the determination of chlorsulfuron, monosulfuron, and thifensulfuron methyl in maize samples.     

Simultaneous determination of thiamphenicol,florfenicol and florfenicol amine in swine muscle by liquid chromatography–tandem mass spectrometry with immunoaffinity chromatography clean-up

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.     

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair

A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 2⁵⁻¹ fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg⁻¹ (CBD and CBN) and 20–500 pg mg⁻¹ (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg⁻¹ and 1–20 pg mg⁻¹, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg⁻¹ (CBD), 20–232 pg mg⁻¹ (THC) and 9–107 pg mg⁻¹ (CBN), confirming the suitability of the method for monitoring studies.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Determination of melatonin in Acyrthosiphon pisum aphids by liquid chromatography–tandem mass spectrometry

Melatonin is a hormone mainly involved in the regulation of circadian and seasonal rhythms in both invertebrates and vertebrates. Despite the identification of melatonin in many insects, its involvement in the insect seasonal response remains unclear. A liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for melatonin analysis in aphids (Acyrthosiphon pisum) for the first time. After comparing two different procedures and five extraction solvents, a sample preparation procedure with a mixture of methanol/water (50:50) was selected for melatonin extraction. The method was validated by analyzing melatonin recovery at three spiked concentrations (5, 50 and 100 pg/mg) and showed satisfactory recoveries (75–110%), and good repeatability, expressed as relative standard deviation (<10%). Limits of detection (LOD) and quantitation (LOQ) were 1 pg/mg and 5 pg/mg, respectively. Eight concentration levels were used for constructing the calibration curves which showed good linearity between LOQ and 200 times LOQ. The validated method was successfully applied to 26 aphid samples demonstrating its usefulness for melatonin determination in insects. This is -to our knowledge- the first identification of melatonin in aphids by LC–MS/MS.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

Using liquid chromatography–tandem mass spectrometry to quantify monohydroxylated metabolites of polycyclic aromatic hydrocarbons in urine

We present an assay which employs enzyme digestion and solid phase extraction followed by liquid chromatography–tandem mass spectrometry to simultaneously quantify 16 hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in 3-ml samples of urine. The analytes consisted of 2-, 3-, and 4-ring OHPAHs, namely, 1- and 2-hydroxynaphthalene (1- and 2-OHNAP), 2-hydroxyfluorine (2-OHFLU), 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene (1-, 2-, 3-, 4-, and 9-OHPHE), 1-hydroxypyrene (1-OHPYR), 1- and 2-hydroxybenzo(a)anthracene (1- and 2-OHBAA), 3- and 6-hydroxychrysene (3- and 6-OHCHR) and 3-, 7-, and 9-hydroxybenzo(a)pyrene (3-, 7-, and 9-OHBAP). The method was validated using urine samples from steel workers and control subjects. The coefficients of variation of the method for the particular analytes were between 7% and 27% and the limits of quantitation were between 0.002 and 0.010 μg/l urine. The 2- and 3-ring OHPAHs were easily quantified in all subjects. However, 1-OHPYR was the only representative of the 4- and 5-ring metabolites that could be quantified. Pairwise correlations showed that all OHPAHs were highly correlated with each other (0.553 ≤ r ≤ 0.910) and with 1-OHPYR (0.614 ≤ r ≤ 0.910), the metabolite most widely accepted as a short-term biomarker of exposure to PAHs. The analyte, 2-OHNAP exhibited the lowest pairwise correlations with the other OHPAHs (0.542 ≤ r ≤ 0.628), presumably due to confounding by smoking. Metabolites of phenanthrene, an abundant PAH and the smallest to possess a bay region, are promising OHPAHs for characterizing both exposures to PAHs and the various metabolic pathways.     

A sensitive and specific assay for glutathione with potential application to glutathione disulphide,using high-performance liquid chromatography–tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS–MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-μl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C₁₈ (150×4.6 mm, 5 μm) column at 35°C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

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.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Enantioselective determination of doxazosin in human plasma by liquid chromatography–tandem mass spectrometry using ovomucoid chiral stationary phase

An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5 mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452 → 344 for doxazosin enantiomers, and m/z 384 → 247 for prazosin (internal standard). The method was linear in the concentration range of 0.100–50.0 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0–11.1% and 5.7–7.6% for R-(−)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4–99.5% for R-(−)-doxazosin and 96.8–102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.     

Measurement of urinary oxypurinol by high performance liquid chromatography–tandem mass spectrometry

Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 μL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 μL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 μm, 100 mm × 2.1 mm, Waters) at 30 °C, using a mobile phase comprised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8 → 108.0; internal standard: m/z 164.9 → 121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10–200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r² > 0.997, n = 7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n = 5) and inter-day (n = 7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1–104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze–thaw cycles and when stored at room temperature for up to 6 h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol.     

Determination of free-form of cocaine in rat brain by liquid chromatography–electrospray mass spectrometry with in vivo microdialysis

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) method with in vivo microdialysis for the determination of free-form of cocaine (COC) in rat brain has been developed. A C₁₈ column and a gradient elution were employed for the separation. The [M+H]⁺ (m/z=304) and a fragmented ion (m/z=182) were detected using positive ion mode detection. Selective ion monitoring was utilized for quantitative measurement. The linearity of this assay was good ranging from 0.01 to 1.0 μM (r²=0.999). The inter- and intra-day precisions showed relative standard deviations ranging from 1.0% to 3.3% and 1.0% to 3.6%, respectively. In addition, the detection of one COC metabolite, benzoylecgonine (BE), by this assay was also investigated. The linearity, precision, and detection limit associated with this method for BE were determined. The application of this newly developed method was demonstrated by examining the pharmacokinetics of COC in rat brain.     

Determination of vinpocetine and its primary metabolite,apovincaminic acid,in rat plasma by liquid chromatography–tandem mass spectrometry

A precise and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of vinpocetine (VP) and its primary metabolite, apovincaminic acid (AVA), in rat plasma was developed and validated. The analytes and the internal standard-dimenhydrinate were extracted from 50 μL aliquots of rat plasma via solid–liquid extraction. Chromatographic separation was achieved in a run time of 3.5 min on a C₁₈ column under isocratic conditions. 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 VP, AVA and IS were m/z 351.4 → 280.2, 323.2 → 280.2 and 256.2 → 167.3 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 0.5–500 ng/mL for both VP and AVA was evaluated with mean correlation coefficient (r) of 0.9970 and 0.9984 respectively. The precision of the assay (RSD%) was less than 8.55% at all concentrations levels for both VP and AVA. This method was successfully applied to a pharmacokinetic study of VP in rats after intravenous (1 mg/kg) and oral (1 mg/kg) administration.