A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis of Caffeine and Theophylline in Human Urine

A weak cation-exchange monolithic column has been prepared in stainless steel tubing and used as the solid-phase extraction material in quantitative analysis of caffeine and theophylline in urine. Column switching, with water as mobile phase, was used for on-line cleaning and screening of human urine samples. Reversed-phase high-performance liquid chromatography was then performed on a C₁₈ column with methanol–water 30:70 (v/v) as mobile phase at a flow rate of 0.5 mL min^?1. Ultraviolet detection was performed at 274 nm. Good linear relationships were obtained between response and concentrations of caffeine and theophylline in the range 0.1–50 μg mL^?1. Absolute recovery ranged from 77.4 to 82.3% and inter-day and intra-day relative standard deviations were less than 5%. The method was suitable for analysis of caffeine and theophylline in human urine, because it eliminated tedious pretreatment and enabled rapid, economic, repeatable, and effective assay of traces of the drugs in biological samples.     

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

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

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

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.     

A Weak Cation-Exchange Monolithic SPE Column for Extraction and Analysis of Caffeine and Theophylline in Human Urine

A weak cation-exchange monolithic column has been prepared in stainless steel tubing and used as the solid-phase extraction material in quantitative analysis of caffeine and theophylline in urine. Column switching, with water as mobile phase, was used for on-line cleaning and screening of human urine samples. Reversed-phase high-performance liquid chromatography was then performed on a C₁₈ column with methanol–water 30:70 (v/v) as mobile phase at a flow rate of 0.5 mL min⁻¹. Ultraviolet detection was performed at 274 nm. Good linear relationships were obtained between response and concentrations of caffeine and theophylline in the range 0.1–50 μg mL⁻¹. Absolute recovery ranged from 77.4 to 82.3% and inter-day and intra-day relative standard deviations were less than 5%. The method was suitable for analysis of caffeine and theophylline in human urine, because it eliminated tedious pretreatment and enabled rapid, economic, repeatable, and effective assay of traces of the drugs in biological samples.

     

Simultaneous Determination of Hydrochlorothiazide, Cilazapril and Its Active Metabolite Cilazaprilat in Urine by Gradient RP-LC

A rapid and simple liquid chromatographic method with UV detection has been developed for the determination of hydrochlorothiazide (HCTZ), cilazapril (CL) and its active metabolite cilazaprilat (CLT) in urine. Sample preparation for urine consisted of solid-phase extraction using styrene-divinylbenzene (SDB-2) cartridges. The chromatographic system was a Zorbax Eclipse XDB-C₁₈ column with a mixture of methanol and 10 mM phosphate buffer, pH 2.3 with gradient (20 to 60% of methanol) as mobile phase at a flow rate of 1.0 mL min^?1. The detection was performed at the wavelength of 206 nm. Enalapril maleat was used as an internal standard. The detector response was linear in the range of 2.4–30.0, 1.6–15.0 and 1.8–20.0 μg mL^?1 for HCTZ, CL and CLT, respectively. LOQ was determined to be 2.4, 1.6 and 1.8 μg mL^?1 for HCTZ, CL and CLT, respectively. Both intra- and inter-day precision were within acceptable limits. The method has been applied to urine samples obtained from three hypertensive patients after intake of HCTZ and CL therapeutic dose.     

A molecular imprint-coated stirrer bar for selective extraction of caffeine, theobromine and theophylline

We have prepared a novel caffeine imprinted polymer on a stir bar that can be used for selective extraction of caffeine, theobromine and theophylline from beverages. The polymerization time and quantities of reagents (template, cross-linker, porogenic solvent) were optimized. The morphology of the molecularly imprinted polymer-coating was studied by scanning electron microscopy and Fourier transform IR spectroscopy. A rapid and sensitive method was worked out for the extraction of caffeine, theobromine and theophylline from beverages by using the molecularly imprinted stir bar followed by HPLC analysis. The effects of extraction solvent, stirring speed, desorption solvent, adsorption and desorption time were optimized. The method displays a linear response in the 5–150 μg L^?1 caffein concentration range, with a correlation coefficient of >0.9904. The recoveries for three analytes in tea, carbonated and functional beverages were 91–108 %, 90–110 % and 93–109 %, with relative standard deviations ranging from 3.6–5.7 %, 3.5–7.9 % and 3.2–7.9 %, respectively.

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.     

Stability-Indicating UPLC Method for the Determination of Bisoprolol Fumarate and Hydrochlorothiazide: Application to Dosage Forms and Biological Sample

A sensitive, selective and accurate ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate and hydrochlorothiazide in their combined dosage forms and as well as in spiked human urine samples. The separation was achieved on an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column, at 40 °C with mobile phase consisting of acetonitrile:phosphate buffer (20 mM) at pH 3.0 with a gradient elution at 225 nm. Bisoprolol fumarate and hydrochlorothiazide were well separated in <1.5 min with good resolution and without any tailing and interference of excipients. The method was fully validated according to ICH guidelines in terms of accuracy, precision, linearity and specificity. A linear response was observed over the concentration range 0.5–150 μg mL^?1 for hydrochlorothiazide and 0.5–250 μg mL^?1 for bisoprolol fumarate. Limit of detection and limit of quantitation for hydrochlorothiazide were calculated as 0.01 and 0.03 μg mL^?1, respectively, and for bisoprolol fumarate were 0.07 and 0.21 μg mL^?1, respectively. Moreover, bisoprolol fumarate and hydrochlorothiazide were subjected to degradation conditions such as hydrolytic, oxidative and thermal stress conditions to evaluate the ability of the proposed method for the separation of bisoprolol fumarate and hydrochlorothiazide from their degradation compounds.     

Capillary GC Analysis of Glyoxal and Methylglyoxal in the Serum and Urine of Diabetic Patients After Use of 2,3-Diamino-2,3-dimethylbutane as Derivatizing Reagent

The dicarbonyl compounds glyoxal, methylglyoxal, and dimethylglyoxal have been separated by capillary GC on a 30 m × 0.32 mm i.d. HP-5 column after precolumn derivatization with 2,3-diamino-2,3-dimethylbutane at pH 4. Chromatographic separation was complete in 6 min. Nitrogen was used as carrier gas at a flow rate of 2 mL min^?1. Split injection was performed with a split ratio of 10:1 (v/v). The derivatives were monitored by flame-ionization detection, and linear calibration plots were obtained in the ranges 0.06–0.69, 0.05–1.01, and 0.07–1.33 μg mL^?1 for glyoxal, methylglyoxal, and dimethylglyoxal, respectively; the respective detection limits were 20, 10, and 10 ng mL^?1. Glyoxal and methylglyoxal were analyzed in serum and urine from diabetics and from healthy volunteers. Amounts of glyoxal and methylglyoxal in serum from diabetic patients were 0.19–0.33 and 0.20–0.29 μg mL^?1, respectively, with respective relative standard deviations (RSD) of 0.8–1.0 and 0.8–1.1%. Amounts of glyoxal and methylglyoxal in serum from healthy volunteers were 0.05–0.08 and 0.04–0.10 μg mL^?1, respectively, with respective RSD of 0.9–1.2 and 1.0–1.2%. Levels of glyoxal and methylglyoxal in urine from diabetic patients were 0.18–0.40 and 0.25–0.36 μg mL^?1, respectively.     

Quantification of moxifloxacin in urine using surface-enhanced Raman spectroscopy (SERS) and multivariate curve resolution on a nanostructured gold surface

A simple procedure is proposed for the determination of the antibiotic moxifloxacin in urine using nanostructured gold as surface-enhanced Raman scattering signal enhancer. The standard addition method in conjunction to multivariate curve resolution-alternating least squares was applied to eliminate the matrix effect and to isolate the spectral contribution of the analyte. Even in the presence of unexpected interferences in the urinary media, it was possible to extract and quantify the analyte response, reaching, in this way, the so-called second-order advantage from first-order data. Moreover, although a saturation phenomenon of the metallic surface was observed, the results of the proposed methodology presented important advantages such as high sensitivity and simpler experimental procedures. The moxifloxacin was determined at levels of 0.70 and 1.50 μg mL^?1 in urine diluted to 1.0 % (corresponding to 70.0 and 150 μg mL^?1 in the original samples) with relative errors of 4.23 and 8.70 %, respectively. The limit of detection (0.085 μg mL^?1) and limit of quantification (0.26 μg mL^?1) values indicated that the quantification can be accomplished in urine up to 24 h after the administration of a single 400-mg dose.     

Determination of Nateglinide in Human Plasma by LC-ESI-MS and Its Application to Bioequivalence Study

To evaluate the bioequivalence of nateglinide, a rapid and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated to determine nateglinide for human plasma samples. The analyte was detected using electrospray positive ionization mass spectrometry in the selected ion monitoring mode. Tinidazole was used as the internal standard. A good linear relationship obtained in the concentration ranged from 0.05 to 16 μg mL^?1 (r ² = 0.9993). Lower limit of quantification was 0.05 μg mL^?1 using 100 μL of plasma sample. Intra- and inter-day relative standard deviations were 2.1–7.5 and 4.7–8.9%, respectively. Among the pharmacokinetic data obtained, T _max was 2.09 ± 1.06 h for reference formulation and 2.40 ± 0.97 h for test formulation. C _max was 4.17 ± 1.31 μg mL^?1 for reference formulation and 4.37 ± 1.53 μg mL^?1 for test formulation. The half-life (t _½) was 1.93 ± 0.44 h for reference formulation and 1.92 ± 0.29 h for test formulation. AUC_0–10h was 13.67 ± 4.36 μg h mL^?1 for reference formulation and 13.21 ± 4.09 μg h mL^?1 for test formulation. This method was successfully applied to the pharmacokinetic study in human plasma samples.     

A Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL^?1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL^?1 and a limit of quantitation at 0.02 μg mL^?1, respectively.     

Determination of trans-Resveratrol in Bio-Matrices for in Vitro, ex Vivo, and in Vivo Pharmacokinetic Studies by LC Spectrometric Analysis

A simple and reproducible liquid chromatographic method was developed for analyzing trans-resveratrol (TR) in cell suspension, intestinal Krebs’ buffer and rat plasma. TR and internal standard (IS, caffeine) were extracted by simple liquid–liquid extraction with acetonitrile. A chromatographic separation of TR and IS was achieved by Hypersil ODS₂ C₁₈ column using the mobile phase consisting of a mixture of methanol and distilled water with approximate retention times of 5.5 and 3.4 min, respectively. The detector wavelength was 303 nm. The limit of quantifications in cell suspension, Krebs’ buffer, and rat plasma were 0.10 μM, 0.05 μg mL^?1, and 0.05 μg mL^?1. The coefficients of correlation were better than 0.9995 in all solvents. The recovery of TR in the three bio-samples ranged from 86.64 to 102.4%. Intra-day and inter-day accuracy were in the range 0.55–11.50%. The proposed method was successfully applied to Caco-2 cells, everted gut sac and rat pharmacokinetic studies. Among the pharmacokinetic data obtained, TR was concentration-dependent uptaken by Caco-2 cells. The colon was the best situation for TR absorption. The absorption of TR after oral administration was rapid, T _1/2 and AUC _0~∞ were 104 min, and 3.49 ± 0.55 min·(μg mL mg)^?1, respectively.     

Analysis of Pazufloxacin Mesilate in Human Plasma and Urine by LC with Fluorescence and UV Detection,and Its Application to Pharmacokinetic Study

A liquid chromatographic method for analysis of pazufloxacin mesilate in human plasma and urine has been developed and validated for selectivity, sensitivity, accuracy, precision, and stability in pharmacokinetic analysis. The sensitivity of the method was 0.02 μg mL^?1 in plasma and 0.5 μg mL^?1 in urine, with overall intra-day and inter-day precision (RSD < 10%) and accuracy (90–120%) acceptable for clinical pharmacokinetic analysis. Recovery from plasma and urine was 80–110% for both pazufloxacin mesilate and enoxacin, the internal standard. Pazufloxacin was stable in both plasma and urine, with no significant degradation under four different conditions. The method was successfully used in a preliminary study of the bioavailability of pazufloxacin mesilate in healthy human volunteers after intravenous administration of 300 and 500 mg.     

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.

     

Simultaneous Determination of Five Major Compounds in Polygonum cuspidatum by HPLC

An RP-HPLC method was developed for the first time to simultaneously determine five major compounds in Polygonum cuspidatum, namely resveratrol, polydatin, anthraglycoside B, emodin and physcion with UV detection at 306 nm. The column was an Agilent Zorbax SB-C₁₈ (250 × 4.6 mm i.d., 5 μm). The separation was carried out with a gradient program. The mobile phase was acetonitrile–water (containing 0.1% formic acid) at a flow rate of 1.0 mL min^?1. The standard curve was rectilinear in the range of 2.04–62.96 μg mL^?1 (r = 0.9998) for resveratrol, 20.13–239.7 μg mL^?1 (r = 0.9998) for polydatin, 7.19–71.92 μg mL^?1 (r = 1.0000) for anthraglycoside B, 2.68–83.68 μg mL^?1 (r = 0.9998) for emodin and 0.60–14.37 μg mL^?1 (r = 0.9997) for physcion. The recoveries of the markers were 96.0, 106.5, 97.8, 97.9 and 98.1%, respectively. The relative standard deviation of intra-day and inter-day were less than 5.0 and 2.3%. This method was simple, accurate and reproducible. The developed method was successfully applied to analyze five compounds in P. cuspidatum of 20 commercial brands.     

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.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.     

Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL^?1 for ascorbic acid, 0.084–0.250 mg mL^?1 for chlorogenic acid, 0.053–0.360 mg mL^?1 for caffeic acid, and 0.016–0.250 mg mL^?1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL^?1 for ascorbic acid, 18 and 54 μg mL^?1 for chlorogenic acid, 37 and 112 μg mL^?1 for caffeic acid, and 11 and 34 μg mL^?1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.