Solid phase extraction--non-aqueous capillary electrophoresis for determination of metformin, phenformin and glyburide in human plasma

Solid phase extraction (SPE) was coupled at line to capillary electrophoresis (CE) for the determination of three basic and neutral diabetic drugs (metformin, phenformin and glyburide) in human plasma. The SPE procedure employed a C(18) cartridge to remove most of the water and proteins from the plasma sample. Analyte detectability was increased due to trace enrichment during the SPE process. Elution of metformin, phenformin and glyburide was achieved with methanol+3% acetic acid. CE analysis was performed using a non-aqueous buffer, acetonitrile+5mM ammonium acetate+5% acetic acid, which afforded rapid separation of metformin from phenformin within 3 min. Glyburide, with a migration time longer than 6 min, did not cause any interference. The present SPE-CE method, with an electrokinetic injection time of 6s and UV detection at 240 nm, was useful for monitoring down to 1 microg/mL of metformin and phenformin in human plasma. When the electrokinetic injection time was increased to 36s, the detection limits were improved to 12 ng/mL for metformin and 6 ng/mL for phenformin.     

Simultaneous determination of glycyrrhizin, a marker component in radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and alpha-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823--> 453 for GLY, 471--> 177 for GA and 752--> 456 for IS. The LC-MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.     

Activation of pyruvate dehydrogenase complex (PDC) of rat heart mitochondria by glyburide

The effects of the second generation sulfonylurea, glyburide, on the pyruvate dehydrogenase multienzyme complex (PDC) of rat myocardial tissue were examined using rat ventricular slices and isolated mitochondria. Therapeutic concentrations (10(-7) to 10(-6)M) of glyburide produced a 30% increase in the decarboxylation of [1(-14)C] pyruvate by the PDC of ventricular tissue. Addition of glyburide to intact rat heart mitochondria stimulated activity of the PDC in a time- and concentration-dependent manner. Half-maximal stimulation of the enzyme occurred with 6 X 10(-5)M glyburide and maximal activation of the enzyme was achieved with 1 X 10(-4)M glyburide. At the height of stimulation, PDC activities were 6-fold greater than those observed under control conditions with succinate alone. When mitochondria were disrupted by sonication or freeze-thawing, glyburide produced no stimulation of pyruvate decarboxylation. We conclude that glyburide directly stimulates the decarboxylation of pyruvate by the PDC of the myocardium. Furthermore, the presence of intact mitochondria is necessary for the stimulatory action of glyburide on the PDC.     

Development and validation of an LC-MS method with electrospray ionization for quantitation of digoxin in human plasma and urine: application to a pharmacokinetic study

A highly sensitive and specific LC-MS method was developed and validated for the quantification of digoxin in human plasma and urine using d5-dihydrodigoxin as internal standard (IS). The assay procedure involved extraction of digoxin and IS from human plasma with chloroform-isopropanol (95:5, v/v). Chromatogrphic separation was achieved on a Spherisorb ODS2 column using a gradient mobile phase with 5 mmol/L ammonium acetate in water with 1% acetic acid and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective [M+K](+) ions, m/z 819.4 for digoxin and m/z 826.4 for IS. The method was proved to be accurate and precise at linearity range of 0.12-19.60 ng/mL in plasma with a correlation coefficient (r(2)) of >or=0.9968 and 1.2-196.0 ng/mL in urine. The limit of quantification achieved with this method was 0.12 ng/mL in plasma and 1.2 ng/mL in urine. The intra- and inter-assay precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers following intravenous administration of digoxin.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

Simultaneous determination of four active alkaloids from a traditional Chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS

A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Automated liquid chromatography-tandem mass spectrometry method for the analysis of firocoxib in urine and plasma from horse and dog

A rugged, sensitive and efficient liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 microL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column. The mobile phase was 45% acetonitrile and 55% of a 2 mM ammonium formate buffer. The method was accurate (88-107%) and precise (CV<12.2%) within and between sets. Extraction efficiencies (recovery)>93% were achieved and ionization efficiencies (due to matrix effects) were >72%. Extensive stability and ruggedness testing was also performed; therefore, the method can be used for pharmacokinetic studies as well as drug monitoring and screening. The data presented here is the first LC-MS/MS method for the quantitation of firocoxib in plasma (LLOQ of 1 ng/mL), a 25-fold improvement in sensitivity over the HPLC-UV method and the first quantitative method for firocoxib in urine (LLOQ of 5 ng/mL). Additionally the sample preparation process has been automated to improve efficiency.     

A sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from Saposhnikovia root in rat plasma and urine

A sensitive and efficient liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C(18) column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0-100 ng/mL in rat plasma and 10-1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5-150 ng/mL in plasma and 15-1500 ng/mL in urine for 4'-O-D-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9% as terms of relative error (R.E.). The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC-MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.     

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.     

Specificity of photolabeling of beta-cell membrane proteins with an 125I-labeled glyburide analog.

The interaction between sulfonylureas and membrane proteins from a hamster insulin-secreting tumor (HIT) cell line has been examined. Four HIT cell membrane proteins were covalently linked to an 125I-labeled glyburide analog by photolabeling. Three photolabeled polypeptides of M(r) 65,000, 55,000, and 30,000 were identified as low affinity "glyburide receptors." These proteins appear to be of similar abundance, when quantitated by photolabeling, with half-maximal displacements (Ki values) by glyburide, glipizide, and tolbutamide in the low micromolar range. The glyburide analog is more tightly bound to a M(r) 140,000 protein with dissociation constants, determined by filtration binding assays and by photolabeling, of 7 and 9.0 nM, respectively. The labeled analog was displaced from the M(r) 140,000 protein by glyburide, glipizide and tolbutamide with Ki values of 3.3 nM, 103 nM, and 25 microM, respectively, as estimated by photolabeling. Optimal conditions established for visualizing the M(r) 140,000 band on autoradiograms prepared after UV cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis include irradiating the radioligand-receptor complex at 1.5 J/cm2 at 312 nm, followed by heating samples in pH 9.0 sodium dodecyl sulfate-gel sample buffer. With receptor sites partially occupied (5 nM radioligand), approximately 0.75% of the protein is photocoupled to the radioligand and visualized by autoradiography. Our results confirm that the M(r) 140,000 polypeptide contains the beta-cell high affinity glyburide binding site and show that the second generation sulfonylurea antidiabetic drugs have a selective increase in affinity for this receptor.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of oseltamivir and its metabolite oseltamivir carboxylate in plasma, saliva and urine

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm x 2.1 mm) using a mobile phase gradient containing acetonitrile-ammonium acetate buffer (pH 3.5; 10mM) at a flow rate of 500 microL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

LC/MS/MS measurement of penicillin G in bovine plasma, urine, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.     

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250mmx4.6mm i.d., 5microm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100mg, 1mL) and needing small amounts of plasma or urine (300microL). The calibration curves were linear over a working range of 10.0-1500.0ng/mL for methadone and of 5.0-500.0ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4ng/mL for methadone and 0.5 and 0.2ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.     

Determination of higenamine in human plasma and urine using liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry

Higenamine is an active ingredient of Aconite root in Chinese herbal medicine and might be used as a new agent for a pharmaceutical stress test and was approved to undergo clinical pharmacokinetic study. Therefore, there exists a need to establish a sensitive and rapid method for the determination of higenamine in human plasma and urine. This paper described a sensitive and rapid method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of higenamine in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the compounds from biological matrices followed by injection of the extracts onto an Atlantis dC18 column with isocratic elution. The mobile phase was 0.05% formic acid in water-methanol (40:60, v/v). The mass spectrometry was carried out using positive electrospray ionization (ESI) and data acquisition was carried out in the multiple reaction monitoring (MRM) mode. The method was fully validated over the concentration range of 0.100-50.0 ng/mL and 1.00-500 ng/mL in plasma and urine, respectively. The lower limits of quantification (LLOQs) were 0.100 and 1.00 ng/mL in plasma and urine, respectively. Inter- and intra-batch precision was less than 15% and the accuracy was within 85-115% for both plasma and urine. Extraction recovery was 82.1% and 56.6% in plasma and urine, respectively. Selectivity, matrix effects and stability were also validated in human plasma and urine. The method was applied to the pharmacokinetic study of higenamine hydrochloride in Chinese healthy subjects.     

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.     

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14C from NaH14CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[1-14C]alanine. Glyburide (20 microM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaH14CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-1,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of tolbutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.     

Biocompatible in-tube solid-phase microextraction coupled to HPLC for the determination of angiotensin II receptor antagonists in human plasma and urine

A poly (methacrylic acid-ethylene glycol dimethacrylate, MAA-EGDMA) monolithic capillary was used for the in-tube solid-phase microextraction (in-tube SPME) of several angiotensin II receptor antagonists (ARA-IIs) from human plasma and urine. Under the optimized extraction condition, the protein component of the biological sample was flushed through the monolithic capillary, while the analytes were successfully trapped. Coupled to HPLC with fluorescence detection, this on-line in-tube SPME method was successfully applied for the determination of candesartan, losartan, irbesartan, valsartan, telmisartan, and their detection limits were found to be 0.1-15.3ng/mL and 0.1-15.2ng/mL in human plasma and urine, respectively. The method was linear over the range of 0.5-200ng/mL for telmisartan, 5-2000ng/mL for candesartan and irbesartan, 10-2000ng/mL for valsartan, and 50-5000ng/mL for losartan with correlation coefficients being above 0.9985 in plasma sample and above 0.9994 in urine sample. The method reproducibility was evaluated at three concentration levels, resulting in the R.S.D. <7%. The poly (MAA-EGDMA) monolithic capillary was demonstrated to be robust and biocompatible by using direct injections of biological samples.     

Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250–2500 and 50–1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.