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.

     

Determination of Pantoprazole, Rabeprazole, Esomeprazole, Domperidone and Itopride in Pharmaceutical Products by Reversed Phase Liquid Chromatography Using Single Mobile Phase

A simple, sensitive, and precise high performance liquid chromatographic method for the analysis of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride, with ultraviolet detection at 210 nm, has been developed, validated, and used for the determination of compounds in commercial pharmaceutical products. The compounds were well separated on a Hypersil BDS C18 reversed-phase column by use of a mobile phase consisting of 0.05 M, 4.70 pH, potassium dihydrogen phosphate buffer - acetonitrile (720:280 v/v) at a flow rate of 1.0 mL min^?1. The linearity ranges were 400–4,000 ng mL^?1 for pantoprazole, 200–2,000 ng mL^?1 for rabeprazole, 400–4,000 ng mL^?1 for esomeprazole, 300–3,000 ng mL^?1 for domperidone and 500–5,000 ng mL^?1 for itopride. Limits of detection (LOD) obtained were: pantoprazole 147.51 ng mL^?1, rabeprazole 65.65 ng mL^?1, esomeprazole 131.27 ng mL^?1, domperidone 98.33 ng mL^?1 and itopride 162.35 ng mL^?1. The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride using single mobile phase.     

Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C₈ reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min^?1. There is good linearity in the range of 0.5–500 ng mL^?1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL^?1 and 0.08 ng mL^?1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL^?1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL^?1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.     

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

Immunofluorescent Analysis with Magnetic Nanoparticles for Simultaneous Determination of Antibiotic Residues in Milk

An analytical system for simultaneous detection of three antibiotic residues in milk (penicillin, tetracycline, and sulfadimethoxine sodium salt) was developed. The method was based on a fluorescence immunoassay. Magnetic nanoparticles (MNPs) were prepared and then coated with 3-(aminopropyl) triethoxysilane (APTES). Antibodies, against penicillin, tetracycline, and sulfadimethoxine, were immobilized on APTES-MNPs. Two immobilization methods were used: a random and an oriented method by protein A. The fluorochrome-antigen conjugates, containing FITC, ATTO 590, and ATTO 630 were obtained. Three separate immunofluorescent analyses were investigated for penicillin, tetracycline, and sulfadimethoxine, first in buffer and then in milk. Linearity values of standard curves in milk were: for penicillin 4–15 ng mL^?1, for tetracycline 50–500 ng mL^?1 (using random immobilization) and 100–500 ng mL^?1(using oriented immobilization), and for sulfadimethoxine 100–500 ng mL^?1. The linearity and sensitivity of calibration curves of determination of penicillin, tetracycline and sulfadimethoxine in milk were very close to the obtained results for separate determination of three antibiotics. It was shown that the multiple immunofluorescence analysis for antibiotic residues in milk without preliminary treatment is effective and rapid.     

LC-MS-MS Method for Simultaneous Determination of Icariin and Its Active Metabolite Icariside II in Human Plasma

An LC-MS-MS method was revised and validated for simultaneous determination of icariin and its active metabolite icariside II in human plasma. The analytes and daidzein (IS) were extracted by liquid–liquid extraction and analyzed by LC-MS-MS. The separation was performed by a Zorbax SB-C₁₈ column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol–water–formic acid (65:35:0.035, v/v/v) at a flow rate of 0.25 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring mode using the electrospray ionization technique in positive mode. The method had lower limits of quantitation 0.2 and 0.1 ng mL^?1 for icariin and icariside II, respectively, using 500 μL plasma sample. The linear calibration curves were obtained in the concentration range of 0.2–100 ng mL^?1 for icariin and 0.1–100 ng mL^?1 for icariside II. The RSD values of intra- and inter-day precision calculated from quality control (QC) samples were less than 7.2% for icariin and less than 6.5% for icariside II. The accuracy as determined from QC samples was within 3.8% for each analyte. The method has been applied to determine and evaluate the pharmacokinetic of icariin and its metabolite icariside II in volunteers following oral administration of icariin and extract of Epimedium, respectively.     

LC–MS–MS Method for Simultaneous Analysis of Abacavir and Lamivudine in Human Plasma, and Its Application to a Bioequivalence Study

A rapid and sensitive LC–MS–MS method has been developed and validated for simultaneous analysis of abacavir (ABA) and lamivudine (LAM) in human plasma. The analytes were extracted from human plasma by SPE. Nelfinavir (NEL) and emtricitabine (EMT) were used as the internal standards for ABA and LAM, respectively. An RP18 column enabled chromatographic separation of the analytes. The method involves simple isocratic chromatography and MS detection in positive-ionization mode. Validation of the method showed response was a linear function of concentration in the ranges 100.0–7000.0 ng mL^?1 for ABA and 80.0–5000.0 ng mL^?1 for LAM. At the LOQ levels, inter-run and intra-run precision were within 5.80 and 3.51%, respectively, for ABA and within 4.68 and 3.16%, respectively, for LAM. Overall recovery for ABA and LAM was 59.32 and 105.18%, respectively. Total elution time was 2 min only, which enabled quantification of more than 200 plasma samples per day. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.     

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.     

An LC–MS–MS Method for the Simultaneous Quantitation of Acyclovir and Valacyclovir in Human Plasma

A simple, sensitive and selective LC–MS–MS method has been developed for the simultaneous determination of acyclovir and valacyclovir in human plasma. Acyclovir and valacyclovir in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of 0.1% formic acid: methanol (30:70% v/v). The method was validated over a linear range of 47–10,255 and 5–1,075 ng mL^?1 for acyclovir and valacyclovir respectively. The LOQs were 47.6 and 5.0 ng mL^?1. The validated method was applied for the quantitation of acyclovir and valacyclovir from plasma samples in a pharmacokinetic study.     

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.     

An Experimental Design Approach to Selecting the Optimum LC Conditions for the Determination of Local Anaesthetics

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anaesthetics: lidocaine, proparacaine, bupivacaine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C₈ (4.6 × 2.5 mm i.d., particle size 5 μm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 mL min^?1. Local anaesthetics detection was performed by UV-Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacaine and oxybuprocaine were 5.74, 9.28, 16.84 and 26.26 min, respectively. HPLC-UV-Vis method was linear in the range of 50–5,000 ng mL^?1 for lidocaine and proparacaine and 100–5,000 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of detection (LOD) was 25 ng mL^?1 for lidocaine, proparacaine and 30 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng mL^?1 for lidocaine, proparacaine and 100 ng mL^?1 for bupivacaine, oxybuprocaine. In intra-day and inter-day precision and accuracy analysis, the relative standard deviation was found to be less than 8%.     

An Accurate Assay for Simultaneous Determination of Irinotecan and Its Active Metabolite SN-38 in Rat Plasma by LC with Fluorescence Detection

An accurate LC method was developed and validated for simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in rat plasma. Plasma samples were pretreated with 0.4 g mL^?1 sodium dodecyl sulfate to inactive the carboxylesterase and avoid the conversion of CPT-11 to SN-38. Chromatographic separation was achieved on a Diamaonsil C₁₈ column using acetonitrile–50 mM phosphate buffered solution (30:70, v/v) at pH 4.0 as the mobile phase with the flow rate of 1 mL min^?1. The linear quantitation ranges for CPT-11 and SN-38 were 5.05–3,030 and 3.15–315 ng mL^?1 with r ² > 0.99, respectively. The lower limit of quantification (LLOQ) was 2.33 ng mL^?1 for CPT-11 and 0.26 ng mL^?1 for SN-38 with intra- and inter-day relative standard deviation of <12% and the accuracy values of >90%. The method was proved to be accurate and sensitive enough and was successfully applied to a pharmacokinetic study of CPT-11 in rats.     

Simultaneous Quantification of Aliskiren, Valsartan and Sitagliptin by LC with Fluorescence Detection: Evidence of Pharmacokinetic Interaction in Rats

A sensitive, precise and simple LC method for the simultaneous quantification of aliskiren, valsartan and sitagliptin in rat plasma has been developed and validated. The chromatographic separation was achieved on a C₁₈ column (250 mm × 4.6 mm, 5 μm) maintained at room temperature, using isocratic elution with acetonitrile/20 mM ammonium acetate buffer (35:65, v/v), pH adjusted to 4.85 with glacial acetic acid, and detected using a fluorescence detector. Liquid–liquid extraction of the aliskiren, valsartan and sitagliptin from the rat plasma with t-butyl methyl ether resulted in their high recoveries. LC calibration curves based on the extracts from the rat plasma were linear in the range of 25–2,000 ng mL^?1 for aliskiren and sitagliptin and 50–4,000 ng mL^?1 for valsartan. The limits of quantification were 25 ng mL^?1 for aliskiren and sitagliptin and 50 ng mL^?1 for valsartan. The precision and accuracy of the method were well within the generally accepted criteria for biomedical analysis. The described method was successfully applied to study the pharmacokinetics of aliskiren, valsartan and sitagliptin following oral administration, individually as well as in combination in Sprague–Dawley rats. The results of the study implied the occurrence of pharmacokinetic interaction upon the co-administration of these three drugs.     

Simultaneous preconcentration and determination of malachite green and fuchsine dyes in seafood and environmental water samples using nano-alumina-based molecular imprinted polymer solid-phase extraction

Molecular imprinted polymer for determination of malachite green (MG) and fuchsine basic (FU) dyes by spectrophotometry has been used, to develop a novel simultaneous extraction and preconcentration method. Molecularly imprinted layer-coated nano-alumina (MIP@Nano-Al₂O₃) as adsorbent was prepared by surface molecular imprinting technique, and characterised by FTIR spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis (EDAX) and thermogravimetric analysis (TGA). The method is based on simultaneous extraction of MG and FU dyes from aqueous solution by using molecularly imprinted polymer and measuring the absorbance at 617 and 546 nm for MG and FU, respectively. Parameters which affect the extraction efficiency such as pH, volume of eluent and amount of adsorbent were investigated and optimised. Linear calibration curves were obtained in the range of 2–750 ng mL^?1 for MG and 1–240 ng mL^?1 for FU under optimum conditions. Detection limit based on three times the standard deviation of the blank (3S_b) was 0.655 and 0.245 ng mL^?1 (n = 10) for MG and FU, respectively. The relative standard deviation (RSD) for 100 ng mL^?1 of MG and FU was 2.35 and 3.06% (n = 7), respectively. The method was applied to the simultaneous determination of the dyes in different seafood and environmental water samples.     

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.     

Determination of Alkylphenols in Water by Dispersive Liquid–Liquid Microextraction Based on Solid Formation without a Disperser

Dispersive liquid–liquid microextraction based on solid formation without a disperser combined with high-performance liquid chromatography has been developed for the determination of 4-tert-butylphenol, 4-n-nonylphenol, and 4-tert-octylphenol. This method is rapid, easy, and uses only 10 µL of a low toxicity organic solvent (1-hexadecanethiol) for the extraction solvent and no disperser solvent. The extraction time and centrifugation time require less than 10 min. The linear range was 1–500 ng mL^?1 for 4-tert-butylphenol, 2–1000 ng mL^?1 for 4-tert-octylphenol, and 5–500 ng mL^?1 for 4-n-nonylphenol with r² ≥ 0.9986. The detection limits were between 0.2 and 1.5 ng mL^?1. The recoveries of lake and river water samples were in the range of 79% to 108%, and the relative standard deviations were 5% to 10%.     

Development of a Sensitive LC Assay with Fluorescence Detection for the Determination of Zearalenone in Rat Serum

A simple and sensitive liquid chromatographic assay with fluorescence detection assay was developed for the determination of zearalenone levels in rat serum. The assay utilized a single liquid–liquid extraction with t-butyl methyl ether and isocratic elution using a mobile phase consisting of acetonitrile and 0.1% triethylamine in distilled water (pH = 6) (50:50, v/v). Linearity was observed over a concentration range from 10 to 1,000 ng mL^?1 (r = 0.9995), with the limit of quantification at 10 ng mL^?1 with 100 μL of rat serum. The validated assay was applied to a pharmacokinetic study in rats.     

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.     

Ion-Pair LC Method for Simultaneous Determination of Aliskiren Hemifumarate,Amlodipine Besylate and Hydrochlorothiazide in Pharmaceuticals

A rapid and precise LC method was developed for the simultaneous determination of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) using acetonitrile:25 mM octane sulfonic acid sodium salt monohydrate in water (60:40 v/v) as the mobile phase. The flow rate was maintained at 1.2 mL min^?1 on a stationary phase composed of Supelco, Discovery^® HS (C18) column (25 cm × 4.6 mm, 5 μm). Isocratic elution was applied throughout the analysis. Detection was carried out at λ _max (232 nm) at ambient temperature. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 32–320, 2–44 and 4–64 μg mL^?1 for ALS, AML and HCZ, respectively. LOD and LOQ were estimated and found to be 0.855 and 2.951 μg mL^?1, respectively, for ALS, 0.061 and 0.202 μg mL^?1, respectively, for AML as well as 0.052 and 0.174 μg mL^?1, respectively, for HCZ. The method was successfully applied for the determination of the three drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in pharmaceutical preparations.     

Simultaneous Determination of Ritonavir and Lopinavir in Human Plasma after Protein Precipitation and LC-MS-MS

A simple, rapid, specific and sensitive method has been developed and validated for simultaneous determination of lopinavir and ritonavir in human plasma. The analytical procedure involves a protein precipitation method using fluoxetine as internal standard Separation was carried out on an Inertsil ODS column using a mobile phase consisting of acetonitrile and 5 mM ammonium acetate buffer. The total run time of analysis was 2.0 min. A linear response function was established for the range of concentrations 50.67–10,008.82 ng mL^?1 for lopinavir and 5.066–1,000.693 ng mL^?1 for ritonavir. The method was successfully applied to an oral bioequivalence study in humans.