A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL^?1 and detect 50 pg mL^?1 of taurine ranging normally between 65 and 179 mmol L^?1 (8–22 μg mL^?1). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.     

Identification of Acepromazine and Its Metabolites in Horse Plasma and Urine by LC–MS/MS and Accurate Mass Measurement

Acepromazine maleate (Sedalin^®) was administered orally to six thoroughbred horses at a dose of 0.15 mg kg⁻¹. Urine and blood samples were collected up to 412 h post-administration. Plasma and urine were hydrolysed; plasma samples were then processed using liquid–liquid extraction and urine samples using solid-phase extraction. A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification for acepromazine of 10 pg mL⁻¹ in plasma and 100 pg mL⁻¹ in urine. Acepromazine, hydroxyethylpromazine, hydroxyacepromazine, hydroxyethylpromazine sulphoxide, hydroxyethylhydroxypromazine, dihydroxyacepromazine and dihydroxyhydroxyethylpromazine were detected in the post-administration samples. The parent drug and its metabolites were identified using a combination of UPLC–MS/MS and accurate mass measurement. Separation of the structural isomers hydroxyethylpromazine sulphoxide and hydroxyethylhydroxypromazine was another significant outcome of this work and demonstrated the advantages to be gained from investing in chromatographic method development.

     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL⁻¹ for ferulic acid and 1.740–348.0 ng·mL⁻¹ for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL⁻¹ for ferulic acid and 1.740 ng·mL⁻¹ for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.

     

Hollow-Fiber-Based LPME as a Reliable Sampling Method for Gas-Chromatographic Determination of Pharmacokinetic Parameters of Valproic Acid in Rat Plasma

A sensitive and simple method based on two-phase liquid-phase microextraction in porous hollow fiber followed by gas chromatography-flame ionization detection was developed for quantification and pharmacokinetic study of valproic acid (VPA, an antiepileptic drug) in rat plasma after oral administration of pure sodium valproate (25 mg kg⁻¹). Some parameters such as type of organic solvent, pH of sample solution, stirring speed, salt addition, extraction time, and volume of sample that affected extraction efficiency of VPA were optimized. Under optimized microextraction conditions, VPA was extracted with 10 μL 1-octanol from 0.5 mL rat plasma previously diluted with 4.5 mL acidified and salinated water (pH 2) using 1-octanoic acid as internal standard. The limit of detection was 17 ng mL⁻¹ with linear response over the concentration range of 50–10,000 ng mL⁻¹ with correlation coefficient higher than 0.998. The developed method was successfully applied to determination of pharmacokinetic parameters such as t _max (peak time in concentration–time profile), C _max (peak concentration in concentration–time profile), t _1/2 (elimination half-life), AUC_0–t (area under the curve for concentration versus time), clearance, and apparent distribution volume in rats following oral administration of VPA.

     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma,and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min⁻¹. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL⁻¹ for wogonin and 10.8–271 ng mL⁻¹ for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL⁻¹ for wogonin and 10.8 ng mL⁻¹ for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.

     

Development and Validation of LC-UV for Simultaneous Estimation of Rosiglitazone and Glimepride in Human Plasma

A simple, sensitive and specific liquid chromatographic method with UV detection (228 nm) was developed for the simultaneous estimation of rosiglitazone and glimepride in human plasma. Rosiglitazone and glimepride were extracted from plasma using liquid–liquid extraction. Separation was achieved with an RP C₁₈ Column using a mixture of phosphate buffer (50 mM) with octane sulfonic acid (10 mM), methanol and acetonitrile as a mobile phase (55:10:35, v/v). pH was adjusted to 7.0. Amlodipine was used as an internal standard (IS). LOD of the method was found to be 20 ng mL⁻¹ for both drugs. Results were linear over the studied range 40.994–2007.556 ng mL⁻¹ for rosiglitazone (r ² ≥ 0.99) and 41.066–2094.84 ng mL⁻¹ for glimepride( r ² ≥ 0.99). The method was found to be simple, selective, precise and reproducible for the estimation of both drugs from spiked human plasma.

     

Simultaneous Determination of Clopidogrel and Its Carboxylic Acid Metabolite (SR26334) in Human Plasma by LC–ESI–MS–MS: Application to the Therapeutic Drug Monitoring of Clopidogrel

A sensitive and specific liquid chromatography-tandem-mass spectrometry method was developed and validated for the simultaneous determination of clopidogrel and its carboxylic acid metabolite (SR26334) in human plasma using nateglinide and pioglitazone as internal standards. Analytes were extracted from 0.50 mL of plasma using diethyl ether–n-hexane (4:1, v/v). Chromatographic separation was performed on a Teknokroma C₁₈ column with a mobile phase of methanol–water (containing 0.1% formic acid) (80:20, v/v) at a flow rate of 0.20 mL min⁻¹ within 5.6 min. Linearity was established over the concentration range of 0.005–5 ng mL⁻¹ for clopidogrel and 20–2,500 ng mL⁻¹ for SR26334. Intra- and inter-batch standard deviations were less than 9.2% and the accuracy of this assay was found to fall within an acceptable range ≤10.0%. The method was successfully applied to the therapeutic drug monitoring of clopidogrel.

     

Fast LC–MS Electrospray Ionization for the Quantification of Digoxin in Human Plasma and Its Application to Bioequivalence Studies

A fast, sensitive and specific liquid chromatography-mass spectrometry method has been developed for quantification of digoxin in human plasma. The method was optimized to bioequivalence studies aiming higher sensitivity and selectivity than previously published methods, in addition to shorter run time allowing high-throughput sample analyses from volunteers. Chromatographic separation was achieved by an RP-18e column hyphenated to an API 5000 mass spectrometer system set at negative electrospray ionization and operating in the MRM mode. Calibration curve was linear over a wide range of concentration (50.0–6000.0 pg mL⁻¹), with the lower limit of quantification at 50.0 pg mL⁻¹ and without interfering peaks at the retention time of digoxin (2.09 min). Dexamethasone was used as internal standard and samples were cleaned up by liquid-liquid extraction obtaining a mean recovery of 73.8%. Validation results confirmed inter-batch accuracy (−8.66 to 5.78%), precision (4.1–10.6%) and stability, in accordance with the U.S. Food and Drug Administration and the Brazilian National Health Surveillance Agency guidelines. The developed analytical method could be successfully applied to a single oral dose (0.25 mg), one-way, randomized, two-sequence, crossover bioequivalence study validating, up to date, the fastest analysis and the most sensitive and specific method already published for digoxin quantification.

     

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

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

     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min⁻¹. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL⁻¹ in rat plasma, with a 1.00 ng mL⁻¹ lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from −2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.

     

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C₁₈ column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL⁻¹ for zofenopril and 2–1,800 ng mL⁻¹ for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.

     

Improved Ultra-Performance LC Determination of Indapamide in Human Plasma

A simple, rapid, sensitive and selective method for the analysis of indapamide in human plasma, utilizing ultra performance liquid chromatography (UPLC), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and the internal standard, sulfamethazine, were isolated from plasma samples by liquid–liquid extraction with diethyl ether. Separation was performed with an Acquity C18 column. The gradient composition of mobile phase was composed of acetonitrile and sodium dihydrogenphosphate buffer (adjusted to pH 3.33 with 85% o-phosphoric acid) at a flow rate of 0.5 mL min⁻¹. The assay exhibited a linear dynamic range of 1–100 ng mL⁻¹ for indapamide in human plasma. The limit of quantification (LOQ) was 1 ng mL⁻¹. The method was successfully applied to the pharmacokinetic and bioequivalence studies of indapamide formulations.

     

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.

     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL⁻¹ in plasma and 0.32–20 μg mL⁻¹ in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.

     

Accuracy Profile Theory for the Validation of an LC–MS-MS Method for the Determination of Risperidone and 9-Hydroxyrisperidone in Human Plasma

A sensitive and specific LC–MS-MS method is described for the simultaneous quantification of risperidone and 9-hydroxyrisperidone in human plasma. After extraction with tert-butyl methyl ether, plasma samples were separated on an Atlantis HILIC Silica C18 column (4.6 × 150 mm, 5 μm)with a mobile phase of ammonium formate buffer (10 mM, pH 4.0)/acetonitrile (40/60, v/v). Detection was by MS-MS. The method was fully validated according to the accuracy profile theory. It is based on β-expectation tolerance interval for the total measurement error which includes trueness and intermediate precision. The measurement uncertainty derived from β-expectation tolerance interval was estimated at each of the validation standards. The linearity fitted well over the range of 0.11–26.75 ng mL⁻¹ for risperidone with an LLOQ of 0.11 ng mL⁻¹, and for 9-hydroxyrisperidone, at a range of 0.15–37.8 ng mL⁻¹ with an LLOQ of 0.15 ng mL⁻¹. The intra- and inter-batch precision of risperidone were <5.71 and 8.22%, respectively. For 9-hydroxyrisperidone, the data were 5.78 and 6.48%. The recoveries were 88.78% (risperidone) and 70.35% (9-hydroxyrisperidone). The developed method was applied to a pharmacokinetic study of risperidone.

     

LC Determination of Ketorolac in Human Plasma and Urine for Application to Pharmacokinetic Studies

A simple, specific and sensitive liquid chromatographic method has been developed for the assay of ketorolac in human plasma and urine. The clean-up of plasma and urine samples were carried out by protein precipitation procedure and liquid–liquid extraction, respectively. Separation was performed by a Waters sunfire C₁₈ reversed-phase column maintained at 35 °C. The mobile phase was a mixture of 0.02 M phosphate buffer (pH adjusted to 4.5 for plasma samples and to 3.5 for urine samples) and acetonitrile (70:30, v/v) at a flow rate of 1.0 mL min⁻¹. The UV detector was set at 315 nm. Nevirapine was used as an internal standard in the assay of urine sample. The method was validated over the concentration range of 0.05–8 and 0.1–10 μg mL⁻¹ for ketorolac in human plasma and urine, respectively. The limits of detection were 0.02 and 0.04 μg mL⁻¹ for plasma and urine estimation at a signal-to-noise ratio of 3. The limits of quantification were 0.05 and 0.1 μg mL⁻¹ for plasma and urine, respectively. The extraction recoveries were found to be 99.3 ± 4.2 and 80.3 ± 3.7% for plasma and urine, respectively. The intra-day and inter-day standard deviations were less than 0.5. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay demonstrated to be applicable for clinical pharmacokinetic studies.

     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL⁻¹ for ibuprofen, 0.001–1.0 μg mL⁻¹ for naproxen, and 0.01–10.0 μg mL⁻¹ for fenbufen. The limits of detection were 0.5 ng mL⁻¹ for ibuprofen, 0.05 ng mL⁻¹ for naproxen and 0.5 ng mL⁻¹ for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.

     

Quantitative Analysis of Fudosteine in Human Plasma Using a Simple Precipitation Step Followed by LC–MS–MS

Fudosteine [(-)-(R)-2-amino-3-(3-hydroxypropylthio) propionic acid] is a synthetic derivative of l-cysteine as a novel expectorant. To facilitate pharmacokinetics studies of fudosteine in man, a sensitive and specific LC–MS–MS method for the quantitative detection of fudosteine in human plasma was developed and validated. The method involved the addition of fosfomycin as internal standards, protein-precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode. The precursor → product ion transitions were monitored at 177.6 → 90.9 and 136.8 → 78.9 for fudosteine and the IS, respectively. The lower limit of quantitation (LLOQ) was 5 ng mL⁻¹. The calibration curves for fudosteine was linear over a concentration range of 0.005–10 µg mL⁻¹. The intra- and inter-day analyses of QC samples at 0.01, 1, 10 µg mL⁻¹ indicated good precision %RSD between 92.00 and 101.02%. The fudosteine was stable in human plasma stored at room temperature for at least 12 h, 4 °C for at least 12 h, −20 °C for at least 20 days, and at least three freeze–thaw cycles procedures. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of fudosteine in human.

     

A Rapid and Sensitive LC–MS Method for Determination of Ezetimibe Concentration in Human Plasma: Application to a Bioequivalence Study

A selective and highly sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for determination of ezetimibe concentrations in human plasma. Ezetimibe was extracted from plasma with ethyl acetate followed by evaporation of the organic layer and, then, reconstitution of the residue in mobile phase before injection to chromatograph. The mobile phase consisted of acetonitrile-ammonium acetate (10 mM, pH 3.0), 75:25 (v/v). An aliquot of 10 μL was chromatographically analyzed on a prepacked Zorbax XDB-ODS C₁₈ column (2.1 × 100 mm, 3.5 micron). Detection of analytes was achieved by mass spectrometry with atmospheric pressure chemical ionization (APCI) interface in the negative ion mode operated under the multiple-reaction monitoring mode (m/z transition: ezetimibe 408–271). Standard curves were linear (r = 0.998) over the wide ezetimibe concentration range of 0.05–30.0 ng mL⁻¹ with acceptable accuracy and precision. The limit of detection was 0.02 ng mL⁻¹. The validated LC–APCI–MS method has been used successfully throughout a bioequivalence study on an ezetimibe generic product in 24 healthy male volunteers.

     

A Simple and Validated LC Method for the Simultaneous Determination of Three Compounds in Mikania laevigata Extracts

The Mikania genus is widely known as guaco and is used to treat fever, rheumatism, influenza and respiratory diseases. This article deals with the simultaneous quantification of three commercially available phenolic markers (o-coumaric acid, coumarin and syringaldehyde) in M. laevigata extracts, through LC-PDA. The validation data show that the method is specific, accurate, precise and robust, and also indicative of the stability of guaco extract. The method was linear, over a range of 1.25–20.0 μg mL⁻¹ for o-coumaric acid, 2.5–40.0 μg mL⁻¹ for coumarin, and 0.25–4.0 μg mL⁻¹ for syringaldehyde. The range of recovery was 94.3–96.4% for all the components, at a level of 100%.