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.     

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.

     

A Simple and Sensitive LC–MS/MS Method for Simultaneous Determination of Temsirolimus and Its Major Metabolite in Human Whole Blood

A simple, fast and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of the concentrations of temsirolimus and its major metabolite, sirolimus, in human whole blood. The blood sample (100 μL) after adding temsirolimus-d7 and sirolimus-d3 internal standards was precipitated with 0.200 mL of methanol/0.300 M zinc sulfate (70/30, v/v), then analyzed by a Shimatzu LC system coupled to a Sciex API-5000 mass spectrometer. The chromatographic separation was carried out on a BDS Hypersil C8 column (50 × 3.0 mm, 5 μm) at 50 °C with a mobile phase composed of methanol/water/formic acid (72/28/0.1) (v/v/v) containing 2.50 mM ammonium acetate. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode. This method was validated from 0.250 to 100 ng mL⁻¹ for temsirolimus and 0.100 to 40.0 ng mL⁻¹ for sirolimus. The lower limits of quantitation were 0.25 ng mL⁻¹ for temsirolimus and 0.1 ng mL⁻¹ for sirolimus. The intra-day and inter-day precisions (CV %) of spiked quality control (QC) samples were less than 10.4 and 9.6 %, respectively. The accuracies as determined by the relative error for QC samples were less than 12.1 % for intra-day and 7.3 % for inter-day. No significant matrix effect was observed. This method has been successfully applied to analyze clinical pharmacokinetic study samples. The assay reproducibility was also demonstrated by using incurred samples.

     

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.

     

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 Abacavir,Efavirenz and Valganciclovir in Human Serum Samples by Isocratic HPLC-DAD Detection

A rapid, sensitive, and specific reverse phase high performance liquid chromatography with diode array detection procedure for the simultaneous determination of abacavir, efavirenz and valganciclovir in spiked human serum is described. Separation was performed on a 5 μm Waters Spherisorb column (250 × 4.6 mm ID) with acetonitrile: methanol:KH₂PO₄ (at pH 5.00) (40:20:40 v/v/v) isocratic elution at a flow rate of 1.0 mL min⁻¹. Calibration curves were constructed in the range of 50–30,000 ng mL⁻¹ for abacavir and efavirenz, and 10–30,000 ngmL⁻¹ for valganciclovir in serum samples. The limit of detection and limit of quantification concentrations of the HPLC method were 3.80 and 12.68 ng mL⁻¹ for abacavir, 2.61 and 8.69 ng mL⁻¹ for efavirenz, 1.30 and 4.32 ng mL⁻¹ for valganciclovir. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in human serum.

     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL⁻¹ for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL⁻¹. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.

     

Development and Validation of an HPLC–UV–Vis Method for the Determination of Proparacaine in Human Aqueous Humour

A sensitive high performance liquid chromatographic method has been developed and validated for the determination of proparacaine in human aqueous humour. The procedure involved extraction of proparacaine from aqueous humour with cyclohexane. The separation was achieved using a Bondesil C₈ (250 × 4.6 mm i.d., particle size 5 μm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH 3.0, 20 mM) (30:70, v/v). Proparacaine and lidocaine (internal standard, IS) detection was performed by UV–Vis detector at 220 nm. The retention times for proparacaine and IS were 12.01 and 5.58 min, respectively. HPLC–UV–Vis method was linear in the range of 75–4,000 ng mL⁻¹. The limit of detection (LOD) was 25 ng mL⁻¹ and the limit of quantification (LOQ) of proparacaine was found to be 75 ng mL⁻¹ (RSD ≤ 15%, n = 6). In intra- and inter-day precision and accuracy analysis, the relative standard deviation was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Recovery of proparacaine from human aqueous humour was 99.98% at 500 ng mL⁻¹. Proparacaine solutions were stable at least 6 months at +4 and −20 °C. Proparacaine levels of aqueous humour in fifteen volunteers’ were in the range of 80.21 and 459.00 ng mL⁻¹. According to system suitability tests and Shewhart’s quality control charts the proparacaine responses were in the acceptance ranges. Developed method was providing a sufficient quality at least over 3 months for determination of proparacaine in human aqueous humour.

     

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.

     

Quantitative Determination of Fexofenadine in Human Plasma by HPLC-MS

A simple, rapid, sensitive and selective LC-MS method was developed and validated for quantification of fexofenadine in human plasma. The LC-MS system was operated under the positive electrospray ionisation mode (ESI). After liquid–liquid extraction, fexofenadine analysis was performed on a C₁₈ column with a mobile phase of acetonitrile: 10 mM ammonium acetate: formic acid, 70:30:0.1 (v/v/v) at a flow rate of 1 mL min⁻¹ by using loratadine as internal standard. The lower limit of quantitation was 3 ng mL⁻¹ for fexofenadine. The assay precision ranged between 1.05 and 12.56% and accuracy ranged between 82.00 and 109.07%. The validated method was successfully used to analyze human plasma samples in bioequivalence studies.

     

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.

     

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.

     

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.

     

Simultaneous Determination of Ebastine and Its Active Metabolite (Carebastine) in Human Plasma Using LC–MS–MS

A sensitive and rapid LC–MS–MS method was developed for the simultaneous determination of ebastine and carebastine in human plasma. Solid-phase extraction was used to isolate the compounds from the biological matrix followed by separation on a Symmetry C18 column under isocratic conditions. The mobile phase was 10 mM ammonium formate in water/acetonitrile (40:60, v/v). Detection was carried out using a triple-quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The method was fully validated over the concentration range of 0.1–10 ng mL^?1 for ebastine and 0.2–200 ng mL^?1 for carebastine in human plasma, respectively. The lower limit of quantification (LLOQ) was 0.1 ng mL^?1 for ebastine and 0.2 ng mL^?1 for carebastine. For ebastine and carebastine inter- and intra-day precision (CV%) and accuracy values were all within ±15% and 85–115%, respectively. The extraction recovery was on average 60.0% for ebastine and 60.3% for carebastine.     

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.

     

TLC Determination of Amitriptyline HCl,Trifluoperazine HCl,Risperidone and Alprazolam in Pharmaceutical Products

A simple, sensitive, and precise thin layer chromatographic (TLC) method for simultaneous analysis of psychopharmacological drugs like amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam in their single dosage forms has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The TLC separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using carbon tetrachloride:acetone:triethylamine (8:2:0.3, v/v/v), as mobile phase. Densitometric measurements of their spots were achieved at 250 nm over the concentration range for amitriptyline HCl (50–1,200 ng spot⁻¹), trifluoperazine HCl (50–1,200 ng spot⁻¹), risperidone (100–2,400 ng spot⁻¹) and alprazolam (25–600 ng spot⁻¹). Limit of detection (LOD) for amitriptyline HCl (20 ng spot⁻¹), trifluoperazine HCl (20 ng spot⁻¹), risperidone (40 ng spot⁻¹) and alprazolam (5 ng spot⁻¹) was obtained. The study showed that TLC was sensitive and selective for determination of amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam using a single mobile phase. This proposed method is able for simultaneous determination of psychopharmacological drugs and also applicable for analysis of pharmaceutical formulations.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

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.     

Determination of Danofloxacin and Marbofloxacin in Milk Samples by Micellar Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, and sensitive analytical method has been developed for the determination of two fluoroquinolones, danofloxacin and marbofloxacin, in bovine milk samples. Separation and quantification were performed by micellar liquid chromatography with fluorescence detection (MLC?FD), using sodium dodecyl sulfate (SDS) as a surfactant. The influence of the principal factors, namely, the micelle concentration, the amount of organic modifier, tail‐reducing agents, the pH, and the temperature were studied. The suitable condition was found to be 75 mM SDS?10 mM phosphate buffer–18 mM tetrabutylammonium bromide/3% (v/v) 1‐propanol at pH 3.0 for the separation of marbofloxacin, danofloxacin, and tosufloxacin (internal standard) in about 20 min. The linear concentration range of application was 1.8–30.0 ng · mL^?1 for danofloxacin and 16–120 ng · mL^?1 for marbofloxacin, and the relative standard deviation ranged between 4.9 and 2.7%. The limit of detection found for danofloxacin was 0.5 ng · mL^?1 and 5 ng · mL^?1 for marbofloxacin. These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in bovine milk. It was applied to check the eventual existence of these compounds above these limits on commercial milk samples. The validation method was completed with spiked milk samples. Recovery levels obtained were 90.3–108.2%.     

Development and Validation of a RP-HPLC–PDA Method for Determination of Curcuminoids in Microemulsions

A sensitive, specific and rapid high-performance liquid chromatography method was developed in an effort to quantify extremely low curcuminoid levels for the future transdermal experiments where the curcuminoids are incorporated with excipients such as microemulsion, liposomes, and micelles. The chromatographic separation was performed using a Symmetry^® C₁₈, 250 × 4.6 mm, 5-μm column, with a mobile phase composed of 5 mM acetonitrile:phosphoric acid (45:55, v/v) at a flow rate of 1.0 mL min⁻¹, it was sensitive with a low limit of quantitation for curcuminoids (0.626 ng mL⁻¹ for curcumin) and good linearity (r ² ≥ 0.999) over the range 1–100 ng mL⁻¹. All the validation data, such as accuracy and precision, were within the required limits from the ICH guideline. The assay method was successfully applied during forced degradation of curcuminoid solutions. The method retained its accuracy and precision when the standard addition technique was applied.