Application of enzymatic probe sonication extraction for the determination of selected veterinary antibiotics and their main metabolites in fish and mussel samples

A new method based on enzymatic probe sonication extraction prior to high-performance liquid chromatography (HPLC) has been developed for the determination of 11 antibiotics (drugs) and the main metabolites of five of them in fish tissue and mussel samples. The analytes belong to four different classes of antibiotics (sulfonamides, tetracyclines, penicillins and amphenicols). The analysed compounds were sulfadiazine (SDI) and N⁴-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and N⁴-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and N⁴-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT).The main factors affecting the extraction efficiency (type of enzyme, type and volume of extractant, ultrasounds power and extraction time) were optimised in tissue of hake (Merluccius merluccius), anchovy (Engraulis encrasicolus), mussel (Mytilus sp.) and wedge sole (Solea solea). The extraction was carried out using an extraction time of 5 min with 5 mL of water and subsequent clean-up with dichloromethane.High-performance liquid chromatography (HPLC) with diode array (DAD) and fluorescence (FLD) detectors was used for the determination of the antibiotics. The separation of the analysed compounds was conducted by means of a Phenomenex^® Gemini C₁₈ (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) guard-column. Analysed drugs were determined using formic acid 0.1% (v/v) in water and acetonitrile in gradient elution mode as mobile phase. The proposed method was also evaluated by a laboratory assay consisting of the determination of the targeted analytes in samples of Cyprinus carpio which had previously administered the antibiotics.     

Determination of Phenolic Acids in Root Exudates of Allelopathic Rice by Solid Phase Extraction-Ion Chromatography with Conductivity Detection

A simple, effective, and green ion chromatography method with conductivity detection was developed for the determination of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid in the root exudates of allelopathic rice. The analytes were well separated within 25 min in an anion exchange column (150 mm × 4.0 mm i.d., 5 µm particle size) with mixtures of 6.4 m mol L^?1 Na₂CO₃ and 2.0 m mol L^?1 NaHCO₃ as the mobile phase at a flow-rate of 0.7 mL min^?1. Detection limits of benzoic acid, cinnamic acid, syringic acid, and p-hydroxybenzoic acid were 0.05, 0.20, 0.50, and 0.05 µg mL^?1, respectively. Intra- and inter-day precision was ≤4.0% and 3.2%, respectively, and the intra- and inter-day accuracy, indicated by relative error, ranged from ?8.0% to 9.0%. The developed method was successfully used to determine phenolic acids in the root exudates of allelopathic rice. The average recoveries of the analytes were between 90.7% and 103.0%.     

Determination of Mycophenolic Acid (MPA) and Its Acyl and Phenol Glucuronide Metabolits Simultaneously in Human Plasma by a Simplified HPLC Method

Abstract

A simple HPLC method with ultraviolet detection for simultaneous determination of Mycophenolic acid (MPA), its phenol glucuronide metabolite (MPAG) and acyl‐MPAG (AcMPAG) in human plasma was established. The plasma samples were prepared with protein‐preciptaing reagent, and the supernatant was eluted on Zorbax column (250 mm×4.6 mm i.d, 5 µm) with 20 mmol/l NaH₂PO₄ buffer (pH 3.0, adjusted with 20% phosphoric acid) and methanol (45:55, v/v) at 304 nm. The column temperature was 45°C, and the flow rate was 1.2 ml/min. The assay was linear within the range of 0.2–50 µg/L for MPA (r=0.9997), 2.8–531 µg/L for MPAG (r=0.9999), and 0.3–24 µg/L for AcMPAG (r=0.9994). Mean absolute recovery of MPA and its metabolites and internal standard was >80%. The average recoveries of MPA, MPAG, and AcMPAG were 94.0–101.4, 98.4–101.9, and 96.1–104.2%, respectively. The RSD of within‐day and between‐day were all lower than 15%. The method described is sensitive, reproducible, and will be useful in TDM or pharmacokinetic studies of MPA.     

Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD-FLD) in human urine samples

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N⁴-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N⁴-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N⁴-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex^® Gemini C₁₈ (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) guard column. Analyzed drugs were determined within 34 min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut^® Plexa™ columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.     

Development and validation of an LC–MS method for the simultaneous determination of sulfadiazine,trimethoprim, and N4-acetyl-sulfadiazine in muscle plus skin of cultured fish

An analytical method for the determination of both sulfadiazine (SDZ) and trimethoprim (TMP), and also N⁴-acetyl-sulfadiazine (AcSDZ), the main metabolite of SDZ, in fish muscle plus skin has been developed and validated. Dapsone was used as internal standard. The method involves extraction of the analytes from fish tissue by pressurized liquid extraction using water as extractant. Sample cleanup was carried out by solid phase extraction using Abselut Nexus cartridges. Target analytes were quantitatively determined by liquid–chromatography mass spectrometry using single ion monitoring. The developed method was validated according to the European Union requirements (decision 2002/657/EC). The limit of detection for SDZ and AcSDZ was 3.0 and 2.5 µg kg^?1 for TMP. The limit of quantification (LOQ) was 10 µg kg^?1 for SDZ and AcSDZ and 7.5 µg kg^?1 for TMP. The recovery experiments carried out included the concentration levels of 0.5, 1 and 1.5 times the MRLs for SDZ and TMP. Concentration levels for AcSDZ were the same as SDZ. The values obtained were higher than 92.0% with coefficient of variation (CV, %) below 8.6%. The precision of the method, calculated as CV (%), ranged from 0.2 to 6.8% and from 0.8 to 8.9% for intra–day and inter–day analysis, respectively. Decision limit (CC_α) was calculated as 104.3, 53.7 and 105.3 µg kg^?1 for SDZ, TMP and AcSDZ, respectively. Detection capability (CC_β) was calculated as 110.0, 58.8 and 109.7 µg kg^?1 for SDZ, TMP and AcSDZ, respectively. “Matrix effect” and “relative matrix effect” were also evaluated. The method was used for the analysis of fish samples purchased from local markets.     

Development and Validation of a UHPLC UV Method for the In-Process Control of Bosentan Monohydrate Synthesis

Bosentan monohydrate (4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrimidin-4-yl]benzene-1-sulfonamide monohydrate) is a dual endothelin receptor antagonist (ERA) applied in the treatment of pulmonary arterial hypertension. To achieve effective process control of the bosentan monohydrate synthesis, it was necessary to develop a selective and not highly time-consuming method for ultra-high performance liquid chromatography (UHPLC). The method is characterized by adequate sensitivity, reproducibility and selectivity for the determination of bosentan monohydrate and related compounds from all synthetic stages. The UHPLC separation was carried out by reversed phase chromatography on the Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a mobile phase composed of solvent A (0.1 %, v/v, acetic acid in water) and solvent B (methanol), in the gradient mode at the flow rate of 0.4 mL min⁻¹. Limits of detection and quantification for the compounds were ≤0.1 µg mL⁻¹ and 0.3 µg mL⁻¹, respectively. The linearity for all related compounds was investigated as in the range for the active pharmaceutical ingredient (API) and as in the range for the in-process control. The developed method was validated according to the current guidelines, proving the suitability of the method for its intended purpose.

     

Determination of Active Ingredients of Origanum Vulgare L. and Its Medicinal Preparations by Capillary Electrophoresis with Electrochemical Detection

Abstract

A method based on capillary electrophoresis with electrochemical detection (CE‐ED) has been developed for the first time for the separation and determination of isovanillic acid, vanillic acid, quercetin, rosmarinic acid, caffeic acid, and protocatechuic acid in Origanum vulgare L. and its medicinal preparations. The effects of working electrode potential, pH level, concentration of running buffer, separation voltage, and injection time on CE‐ED were investigated. Under the optimum conditions, the analytes could be separated in a 50 mmol L^?1 borate buffer (pH 8.7) within 21 min. A 300‐µm diameter carbon disk electrode has a good response at +0.95 V (vs. SCE) for all analytes. The response was linear over three orders of magnitude with detection limits (S/N=3) ranging from 4×10^?8 g mL^?1 to 2×10^?7 g mL^?1 for the analytes. The method has been successfully applied to the analysis of real sample, with satisfactory results.     

An LC Method for the Determination of Bupropion and Its Main Metabolite,Hydroxybupropion in Human Plasma

A new LC method has been developed and validated for the direct determination of bupropion and its main metabolite, hydroxybupropion in human plasma. Plasma samples were analyzed after a simple, one step protein precipitation with trichloroacetic acid using a C₈ column and mobile phase, consisting of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (40:10:50, v/v/v) and 20 mM 1-heptane sulfonic acid sodium salt with carbamazepine as the internal standard. UV detection was performed at 214 and 254 nm. The method was validated over the concentration range of 60–2,400 and 150–4,700 ng mL⁻¹ for bupropion and hydroxybupropion, respectively. The intra- and inter-day assay variability was less than 15% for the two analytes. Limit of detection values were 24.8 and 63.4 ng mL⁻¹ for bupropion and hydroxybupropion, respectively. The method developed was applied to quantification of bupropion and hydroxybupropion in human plasma.

     

Development of a green effervescence-assisted dispersive liquid–liquid microextraction method using a home-made tablet disperser for trace analysis of Cd(II) and Pb(II)

An analytical approach for the determination of trace amounts of Cd(II) and Pb(II) has been developed using a home-made tablet-based effervescence-assisted dispersive liquid–liquid microextraction (DLLME) method which was performed in a narrow-bore tube, followed by flame atomic absorption spectrometry. In this method, a mixture of tartaric acid, sodium bicarbonate and NaCl was used to make the disperser tablet. Then, microlitre level of an extraction solvent was added in the tablet, and then, it was released into a narrow-bore tube containing sample solution and a complexing agent. An acid–base reaction immediately occurred between tartaric acid and sodium bicarbonate, and the produced CO₂ led to the dispersion of the extraction solvent into the solution as tiny droplets and subsequent extraction of the analytes. The method made possible the determination of Cd(II) and Pb(II) in the ranges of 0.1–10 and 1.0–20 µg L^?1, respectively. The limits of detection were obtained 0.43 and 0.05 µg L^?1 for Pb(II) and Cd(II), respectively. The limits of quantifications were 0.80 and 0.09 µg L^?1 for Pb(II) and Cd(II), respectively. Repeatability of the method, which is expressed as relative standard deviation, was obtained 3.1% (n = 6, C = 2 µg L^?1) and 1.3% (n = 6, C = 0.2 µg L^?1) for Pb(II) and Cd(II), respectively. The accuracy of the developed method was verified by analysing a certified reference material, namely SPS-WW2 Batch 108. Relative recoveries (84–107%, obtained at three fortification levels) confirmed the usefulness of the method for analysis of the analytes in the environmental water samples and fruit juices. The method was shown to be fast, reliable and environmentally friendly with low organic solvent consumption.     

Quantitative Analysis of Busulfan in Human Plasma by LC-MS–MS

High-performance liquid chromatography with tandem mass spectrometry has been used for rapid, specific, and sensitive analysis of busulfan in human plasma. Busulfan-d₈ was used as internal standard. Analysis was performed on a C₁₈ column (50 mm × 2.1 mm, 3.5-µm particles) with water–methanol 80:20 (v/v) as mobile phase at a flow-rate of 0.30 mL min⁻¹. Detection was by tandem triple–quadrupole mass spectrometry with turbo ion-spray ionization. Linear calibration plots were obtained over the concentration range 1.096–1,096 ng mL⁻¹. The assay is ideally suited to monitoring of busulfan and determination of its pharmacokinetic data.

     

Rapid LC-APCI-MS-MS Method for Simultaneous Determination of Phenacetin and Its Metabolite Paracetamol in Rabbit Plasma

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C₁₈ column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/v) at a flow of 0.4 mL min^?1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL^?1 for phenacetin and 3–2,000 ng mL^?1 for paracetamol, with a lower limit of quantitation of 4 ng mL^?1 for phenacetin and 3 ng mL^?1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study.     

Simultaneous kinetic–spectrophotometric determination of mycophenolate mofetil and mycophenolic acid based on complexation with Fe(III) using chemometric techniques

Determination of pharmaceutical analytes has been subjected to many investigations, especially in transplantations in which accurate and precise detection of drugs is of importance. In this study, a simple and fast complexation reaction has been employed for simultaneous kinetic–spectrophotometric determination of two immunosuppressant drugs, mycophenolate mofetil and its active metabolite mycophenolic acid, which is based on the reaction between drugs and Fe(III) ions in the presence of sodium dodecyl sulfate as anionic surfactant by standard addition method. The effect of influential parameters including type of surfactant, concentration of Fe(III) ions and pH of the solution on the complexation reaction has been studied, and SDS was chosen as suitable surfactant, while reaction proceeds with 0.1 M Fe(III) at pH 4. Multivariate curve resolution-alternating least squares has been employed for analyzing the multiset data obtained from augmentation of resulting standard addition matrices. Values for limit of detection of method have been calculated as 4.88 and 1.62 µg mL^?1 for mycophenolic acid and mycophenolate mofetil, respectively, and Beer’s law is obeyed over the concentration ranges 10–200 µg mL^?1 for MPM and 50–250 µg mL^?1 for MPA. The proposed method was successfully applied for determination of drugs in plasma serum samples. The accuracy and reliability of the method was further ascertained by recovery studies via standard addition procedure.     

Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate

Abiraterone acetate is a prodrug of Abiraterone widely used for the treatment of metastatic castration resistant prostate cancer. In this study, a simple, sensitive, and rapid stability-indicating reverse phase HPLC method was developed and validated for the determination of Abiraterone acetate in bulk and its pharmaceutical formulation. The method was developed by HPLC using a Hypersil ODS C-18 (150 mm × 4.6 mm, 5 µm) column in a isocratic mode with mobile phase constituted by potassium phosphate buffer and acetonitrile (40:60, v/v%) flow rate was 1.0 mL min^?1, column temperature of 30°C, UV detection wavelength 235 nm, and injection volume of 20 µL. The validated parameters were in accordance with FDA and ICH specifications, assay exhibited a linear range of 25–250 µg mL^?1 with regression (r²) coefficient 0.9998. The limits of detection and quantification were 0.23 and 0.70 µg mL. Accuracy was between 99.34 and 100.07%. The drug was subjected to various stress conditions like acidic, base hydrolysis, oxidation, thermal, and photolytic degradation. Stress study Abiraterone acetate was found susceptible to degrade under hydrolytic (acid and base) conditions. The proposed method has stability indicating the resolution of the main peak from their degradation peaks. The validated method is suitable for quality control application and reduced analysis time.     

Simultaneous Determination of Carbamazepine and its Active Metabolite Carbamazepine-10,11-epoxide in Human Plasma by UPLC

A simple method for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide by ultra performance liquid chromatography (UPLC) with ultraviolet absorbance detection (TUV) was developed. The method involves a two-step protein precipitation by liquid–liquid extraction. Phenytoin sodium was used as the internal standard. The separation was carried out on Acquity C18 column with acetonitrile:methanol:KH₂PO₄ buffer (adjusting pH to 4.6 with 85% o-phosphoric acid) (180/180/170, v/v/v) as the mobile phase at a flow rate of 0.4 mL min⁻¹. Linear detection response was obtained for concentrations ranging from 50 to 5,000 ng mL⁻¹. The limit of quantification (LOQ) was 50 ng mL⁻¹. The method was validated successfully for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide, which can be applied through pharmacokinetics and bioequivalence studies.

     

LC–MS–MS Separation and Simultaneous Determination of Tolterodine and its Active Metabolite, 5-Hydroxymethyl Tolterodine in Human Plasma

A selective, sensitive and high throughput LC–MS–MS method has been developed and validated for the chromatographic separation and quantitation of tolterodine (TOL) and its metabolite 5-hydroxymethyl TOL in human plasma. Sample clean-up concerned liquid–liquid extraction of the drug, metabolite and their respective labelled internal standards from 300 μL human plasma. Both the analytes were chromatographically separated on a Symmetry C18 (100 mm × 4.6 mm, 5 μm particle size) analytical column using 10 mM ammonium formate (pH 5.0 ± 0.1, adjusted with formic acid) and acetonitrile (35:65, v/v) as the mobile phase with a resolution factor of 2.72. The method was validated over the concentration range of 0.025–10.0 ng mL^?1 for both analytes. The process efficiency found for TOL and its metabolite was 98.3 and 99.5%, respectively. The method was successfully applied to a pivotal bioequivalence study in 41 healthy human subjects after oral administration of a 2 mg tablet formulation under fasting conditions.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

An LC Method for the Determination of Bupropion and Its Main Metabolite, Hydroxybupropion in Human Plasma

A new LC method has been developed and validated for the direct determination of bupropion and its main metabolite, hydroxybupropion in human plasma. Plasma samples were analyzed after a simple, one step protein precipitation with trichloroacetic acid using a C₈ column and mobile phase, consisting of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (40:10:50, v/v/v) and 20 mM 1-heptane sulfonic acid sodium salt with carbamazepine as the internal standard. UV detection was performed at 214 and 254 nm. The method was validated over the concentration range of 60–2,400 and 150–4,700 ng mL^?1 for bupropion and hydroxybupropion, respectively. The intra- and inter-day assay variability was less than 15% for the two analytes. Limit of detection values were 24.8 and 63.4 ng mL^?1 for bupropion and hydroxybupropion, respectively. The method developed was applied to quantification of bupropion and hydroxybupropion in human plasma.     

Flow‐Injection Spectrophotometric Determination of N‐acetylcysteine in Pharmaceutical Formulations with On‐Line Solid‐Phase Reactor Containing Zn(II) Phosphate Immobilized in a Polyester Resin

Abstract

A flow‐injection spectrophotometric procedure was developed for determining N‐acetylcysteine in pharmaceutical formulations. The sample was dissolved in deionized water and 400 µl of the solution was injected into a carrier stream of 1.0×10^?2 mol l^?1 sodium borate solution. The sample flowed through a column (70 mm length×2.0 mm i.d.) packed with Zn₃(PO₄)₂ immobilized in a polymeric matrix of polyester resin and Zn(II) ions were released from the solid‐phase reactor because of the formation of the Zn(II) (N‐acetylcysteine)₂ complex. The mixture merged with a stream of borate buffer solution (pH 9.0) containing 5.0×10^?4 mol l^?1 Alizarin red S and the Zn(II)Alizarin red complex formed was measured spectrophotometrically at 540 nm. The analytical curve was linear in the N‐acetylcysteine concentration range from 3.0×10^?5 to 1.5×10^?4 mol l^?1 (4.9 to 24.5 µg ml^?1) with a detections limit of 8.0×10^?6 mol l^?1 (1.3 µg ml^?1). The relative standard deviations (RSDs) were smaller than 0.5% (n=10) for solutions containing 5.0×10^?5 mol l^?1 (8.0 µg ml^?1) and 8.0×10^?5 mol l^?1 (13.0 µg ml^?1) of N‐acetylcysteine, and the analytical frequency was 60 determinations per hour. A paired t‐test showed that all results obtained for N‐acetylcysteine in commercial formulations using the proposed flow‐injection procedure and a comparative procedure agreed at the 95% confidence level.     

Determination of a novel nonfluorinated quinolone,nemonoxacin, in human feces and its glucuronide conjugate in human urine and feces by high‐performance liquid chromatography–triple quadrupole mass spectrometry

Three methods were developed and validated for determination of nemonoxacin in human feces and its major metabolite, nemonoxacin acyl‐β‐ d ‐glucuronide, in human urine and feces. Nemonoxacin was extracted by liquid–liquid extraction in feces homogenate samples and nemonoxacin acyl‐β‐ d ‐glucuronide by a solid‐phase extraction procedure for pretreatment of both urine and feces homogenate sample. Separation was performed on a C₁₈ reversed‐phase column under isocratic elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. Both analytes were determined by liquid chromatography–tandem mass spectrometry with positive electrospray ionization in selected reaction monitoring mode and gatifloxacin as the internal standard. The lower limit of quantitation (LLOQ) of nemonoxacin in feces was 0.12 µg/g and the calibration curve was linear in the concentration range of 0.12–48.00 µg/g. The LLOQ of the metabolite was 0.0010 µg/mL and 0.03 µg/g in urine and feces matrices, while the linear range was 0.0010–0.2000 µg/mL and 0.03–3.00 µg/g, respectively. Validation included selectivity, accuracy, precision, linearity, recovery, matrix effect, carryover, dilution integrity and stability, indicating that the methods can quantify the corresponding analytes with excellent reliability. The validated methods were successfully applied to an absolute bioavailability clinical study of nemonoxacin malate capsule. Copyright © 2014 John Wiley & Sons, Ltd.     

Study on Pharmacokinetics of Liguzinediol and Four Metabolites in Rats by UFLC–MS/MS

A rapid and sensitive UFLC–MS/MS method was developed for the simultaneous determination of liguzinediol and its four primary metabolites (M1, M2, M3, and M4) in rat plasma using antipyrine as internal standards. The analytes were separated on an XR-ODS column (50 mm × 2.0 mm, 2.2 μm) using 0.1 % formic acid–methanol gradient elution at a flow rate of 0.5 mL·min⁻¹. The detection was performed on a triple quadrupole tandem mass spectrometer in a multiple reaction monitoring mode with positive electrospray ionization. Method validation was performed as per the Food and Drug Administration guidelines. The resulting calibration curves offered satisfactory linearity (r > 0.9993) with the set ranges. The limits of quantification for liguzinediol, M1, M2, M3, and M4 were 20, 20, 21, 27, and 10 ng mL⁻¹, respectively. The recovery rates in different matrices ranged from 91.2 to 114.1 %, and the inter-day and intra-day precisions were all less than 13.4 % for the target analytes. After validation, this method was successfully applied to further study pharmacokinetics profiles of liguzinediol and its metabolites after intravenous administration of 10 mg·kg⁻¹ in male and female rats.