Simultaneous Determination of Four Active Components in a Compound Formulation by Liquid Chromatography

Summary A rapid and accurate LC method is described for simultaneous determination of pseudoephedrine hydrochloride (PSE), acetaminophen (AMP), dextromethorphen hydrobromide (DEX), and diphenhydramine hydrochloride (DPH) in a compound formulation. Chromatographic separation of the four drugs was achieved on a Hypersil CN column (150 mm × 4.6 mm, 5 m particle) by use of a mobile phase comprising a mixture of 3 mM ion-pairing solution, 2% aqueous triethylamine solution, and 2 M phosphoric acid, 68:48:88 (v/v), pH 3.0, delivered at 1.0 mL min^–1. Compounds were detected at 215 nm and the run time was less than 10 min. The linearity, accuracy, and precision of the method were found to be acceptable over the concentration ranges 6.1–36.4 g mL^–1 for PSE, 65.0–390.0 g mL^–1 for AMP, 3.1–18.6 g mL^–1 for DEX, and 5.0–30.0 g mL^–1 for DPH.     

RP-HPLC Determination of Famotidine and its Potential Impurities in Pharmaceuticals

A simple, sensitive, and rapid reversed-phase high-performance liquid chromatographic method has been developed for determination of famotidine (FMT) and its impurities in pharmaceutical formulations. Separations were performed on a Supelcosil LC18 column with an isocratic mobile phase—13:87 (v/v) acetonitrile–0.1 M dihydrogen phosphate buffer containing 0.2% triethylamine (pH 3.0). The mobile phase flow rate was 1 mL min^–1 and the detection wavelength was 265 nm. Response was linearly dependent on concentration between 1 and 80 g mL^–1 (regression coefficient, R², from 0.9981 to 0.9999). RSD from determination of method repeatability (intraday) and reproducibility (interday) were <2% (n=6). Lowest detectable concentrations ranged from 0.08 to 0.14 g mL^–1. The proposed liquid chromatographic method can be satisfactorily used for routine quality control of famotidine in pharmaceutical formulations.     

Determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol by postcolumn derivatization witho-phthalaldehyde/2-mercapto-ethanol after ion-pair chromatography

Summary A high-performance liquid chromatographic method is discribed for the determination of 6-amino-2,2-dimethyl-1,3-dioxepan-5-ol using Spherisorb ODS II stationary phase and mobile phase 30:70 (v/v) methanol: aqueous 1-octane sulfonic acid. Detection was fluorimetric following postcolumn derivatization with o-phthaladehyde/2-mercaptoethanol. The procedure was applied to the analysis of aqueous solutions and microcrystalline suspensions in liquid paraffin, prepared for investigation of the toxicological profile. The method was validated for selectivity, linearity of detector response, repeatability, limit of detection and quantitation. The HPLC method was selective. The instrumental limit of detection was 0.5 ng per injection (0.05 g mL^–1). The method detection limits were 0.5 g mL^–1 aqueous solution and 5 g mL^–1 liquid paraffin suspension, the quantitation limit 0.05 mg mL^–1 aqueous solution and 1.0 mg mL^–1 liquid paraffin. Linearity was within 0.94–47.1 g mL^–1. Intra-assay accuracy accounted for 99–100% in the range 0.05–226 mg mL^–1 aqueous solution, intra-assay precision for 2% (C.V.). For microcrystalline liquid paraffin suspensions with 1 and 250 mg mL^–1 99 and 109% was found for intra-assay accuracy. Intra-assay precision was 5% (C.V.). Reliable results over a wide concentration range can be obtained. The procedure is considered valid for determination of the analyte in aqueous solution or microcrystalline paraffin oil suspensions.     

LC Method for the Determination of Multiple Components in a Compound Cold Formulation

An isocratic liquid chromatographic method for determination of acetaminophen (AMP), caffeine (CAF), chlorphenamine maleate (CPM) and guaiacol glyceryl ether (GGE) in a compound cold formulation is described. Separation and quantitation were achieved on a Diamonsil C18 column using a binary mixture of methanol and 1.5% aqueous acetic acid (55: 45, v/v, pH 3.6) as mobile phase delivered at 0.4 mL min^–1. Single wavelength detection was at 220 nm for all four drugs and the run time was < 10 min. The linearity, accuracy and precision of the method were found to be acceptable over the concentration ranges: 16.0–127.8 g mL^–1 for AMP, 6.0–48.2 g mL^–1 for CAF, 5.0–40.0 g mL^–1 for CPM and 10.1–80.6 g mL^–1 for GGE.     

HPLC Determination and Pharmacokinetic Study of Valdecoxib in Human Plasma

A sensitive, simple, and accurate high-performance liquid chromatographic method has been developed for determination of valdecoxib and the internal standard rofecoxib in human plasma. Protein was precipitated from plasma samples by addition of perchloric acid (HClO₄); the drug was then extracted with diethyl ether. Separation was performed on a Cosmosil C₁₈ column (150 mm × 4.6 mm i.d., 5 m particles) with ammonium acetate buffer-acetonitrile, 60:40 (v/v), containing 0.1% TEA, pH 6.5, as mobile phase. Detection and quantification were performed by UV-visible detection at 239 nm. Detection and quantification limits were 3 and 5 ng mL^–1, respectively. The linear concentration range for valdecoxib was 5–400 ng mL^–1. The validated RP HPLC method was used for determination of the pharmacokinetic data for the drug in humans.     

Determination of fosfomycin in human urine by capillary gas chromatography: Application to clinical pharmacokinetic studies

Summary A capillary gas chromatographic method for the determination of fosfomycin in human urine is described. After dilution of the sample and derivatization, analysis was on a HP-1 capillary column and a flame ionization detector was used to determine the bistrimethylsilyl derivative of fosfomycin. Response was linear in the range 50–5000 g mL^–1. The detection limit was about 10 g mL^–1. The within and between day coefficients of variation did not exceed 6%. The method was applied to the determination of fosfomycin in urine samples collected during clinical pharmacokinetic studies.     

HPLC Determination of Ritodrine Hydrochloride in Pharmaceutical Formulations

A simple, rapid, and accurate HPLC method is described for the determination of ritodrine hydrochloride (RTH) in both pure form and pharmaceutical formulations. A Hypersil Shendon ODS column with a mobile phase of dibasic phosphate buffer and acetonitrile (75 : 25) and isoxsuprine hydrochloride were used as an internal standard. The flow rate was 1 mL min^–1 and the effluent was monitored at 270 nm pH 4.0. The calibration graph is linear in the range 2–30 g mL^–1. The proposed HPLC method has been successfully employed for the determination of RTH in Yutopar tablets and injection solutions.     

Development and validation of a novel HPLC/ELSD method for the direct determination of tobramycin in pharmaceuticals,plasma, and urine

A novel method for the direct determination of the aminoglycoside tobramycin was developed and validated based on reversed-phase high-performance liquid chromatography (RP-HPLC) with evaporative light scattering detector (ELSD). Using a Waters ODS-2 C18 Spherisorb column with an evaporation temperature of 45°C and nitrogen pressure of 3.5 bar, the selected mobile phase consisted of water/acetonitrile 55:45 containing 1.5 mL L^–1 HFBA (11.6 mM) in an isocratic mode at a rate of 1.0 mL min^–1. Tobramycins retention time was 4.3 min with an asymmetry factor of 1.7. A logarithmic calibration curve was obtained from 1 to 38 g mL^–1 (r > 0.9998). LOD was 0.3 g mL^–1; within-day %RSD was 1.0 (n = 3, 4.7 g mL^–1) and between-day %RSD was 1.1 (3 days within a week). The developed method was applied to the determination of tobramycin in a pharmaceutical crude substance and formulations (eye drops and ointments). Dilution experiments revealed the absence of interference from excipients (no constant and proportional errors); recovery from spiked samples was 99–103% with %RSD < 2.2 (n = 3×3). The developed HPLC/ELSD method was also found to be applicable in the determination of tobramycin in human plasma (0.6–12.5 g mL^–1) and urine (1.5–12.5 g mL^–1) after solid-phase extraction using carboxylate cartridges followed by solvent evaporation (×2 preconcentration). A mean recovery of 86% for plasma and 91% for urine was obtained.     

LC-APCI-MS-MS methodology for determination of glybenclamide in human plasma

A liquid chromatographic/atmospheric pressure chemical ionization tandem mass spectrometric method (LC-APCI-MS-MS) for the determination of glybenclamide in human plasma is described. Glypizide, an analogue of glybenclamide, was used as internal standard. The analyte was extracted from plasma with diethyl ether/dichloromethane (70:30 v/v). The chromatography uses C₁₈ and 0.01 mol L^–1 acetic acid/acetonitrile (20:80 v/v) as stationary and mobile phase, respectively. Quantitation was preformed by using multiple reaction monitoring (MRM) of the precursor ion (m/z 494.2368.8) and the related product ion (m/z 446.0347.3) using the internal standard method. The analytical curve was linear in the range 1–300 ng mL^–1, and for a 400-L sample of human plasma, the limit of determination of the method was 1 ng mL^–1. The coefficients of variation of the method for intra-assay (within-run precision) and inter-assay (between-run precision) were less than 10%. The method was shown to be suitable for pharmacokinetic studies.An erratum to this article can be found at     

Determination of pKa values of some antihypertensive drugs by liquid chromatography and simultaneous assay of lercanidipine and enalapril in their binary mixtures

In this study, pK_a values were determined using the dependence of the retention factor on the pH of the mobile phase for three ionizable substances, namely, enalapril, lercanidipine and ramipril (IS). The effect of the mobile phase composition on the ionization constant was studied by measuring the pK_a at different methanol-water mixtures, ranging between 50 and 65% (v/v), using LC-DAD method. Two simple, accurate, precise and fully validated analytical methods for the simultaneous determination of enalapril and lercanidipine in combined dosage forms have been developed. Separation was performed on an X-Terra RP-18 column (250 mm × 4.60 mm ID × 5 μm) at 40 °C with the mobile phase of methanol-water 55:45 (v/v) adjusted to pH 2.7 with 15 mM orthophosphoric acid. Isocratic elution was performed in less than 12 min with a flow rate of 1.2 mL min⁻¹. Good sensitivity for the analytes was observed with DAD detection. The LC method allowed quantitation over the 0.50-20.00 μg mL⁻¹ range for enalapril and lercanidipine. The second method depends on first derivative of the ratio-spectra by measurements of the amplitudes at 219.7 nm for enalapril and 233.0 nm for lercanidipine. Calibration graphs were established for 1-20 μg mL⁻¹ for enalapril and 1-16 μg mL⁻¹ lercanidipine, using first derivative of the ratio spectrophotometric method. Both methods have been extensively validated. These methods allow a number of cost and time saving benefits. The described methods can be readily utilized for analysis of pharmaceutical formulations. The methods have been applied, without any interference from excipients, for the simultaneous determination of these compounds in tablets. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations.     

Fluorometric Determination of Nitrogen Dioxide in Atmosphere Sampled by the Liquid Droplet Technique

A new, simple, sensitive and selective fluorometric method for the determination of nitrite has been developed. The reaction of nitrite with hydralazine in acidic medium, heated on a boiling water-bath for 15 min, produced a tetrazolo (5,1-a) phthalazine (Tetra-P). The product formed was measured at _ex = 274 nm and _em = 345 nm. The fluorescence intensity was valid over a nitrite concentration range 0.067–60.3 ng mL^–1, with a detection limit of 0.0091 ng mL^–1. The reproducibility of the proposed method was determined by running a different concentration of nitrite, 13.4, 33.5, and 46.9 ng mL^–1. The % recoveries and the relative standard deviations were found to be 100.6 ± 0.9, 99.9 ± 0.5, and 99.4 ± 1.1%, respectively. The proposed method was applied successfully to the determination of nitrogen dioxide sampled from the atmosphere using the liquid droplet method. The nitrogen dioxide our wind tunnel was controlled by an NO _x analyzer based on a chemiluminescence analyzer detector (CLAD 1000). A linear graph was obtained for the nitrogen dioxide in the wind tunnel vs. NO₂ sampled by the liquid droplet method. The effect of interference substances in the determination showed that cations and anions did not disturb the process. The results obtained were satisfactory when compared with the reference method.     

Determination of the Enantiomers of Tamsulosin Hydrochloride and its Synthetic Intermediates by Chiral Liquid Chromatography

A chiral liquid chromatographic method is described for the determination of the enantiomers of tamsulosin hydrochloride and its synthetic intermediates. Enantioseparation was achieved on a Chiralcel OD-R column (250 mm × 4.6 mm, 10 m) using a mobile phase consisting of a mixture of 0.5 mol L^–1 sodium perchlorate and acetonitrile (80:20, v/v, pH 4.0). The flow rate was 0.4 mL min^–1 and detection was at 223 nm. Excellent enantiomer separations were achieved for tamsulosin hydrochloride and its synthetic intermediates. No other methods are available for the separation of these enantiomers. The method developed in this study has been successfully applied for purity control.     

Kinetic–Spectrophotometric Determination of Trace Silver(I) Using Its Catalytic Effect on the Oxidation Reaction of Fuchsin by Peroxodisulfate in the Presence of 1,10-Phenanthroline As an Activator

A kinetic spectrophotometric method for the determination of trace amounts of Ag(I) in the range of 2–20 ng mL^–1 is reported. The method is based on the catalytic effect of Ag(I) on the oxidation reaction of fuschin by potassium peroxodisulfate in the presence of 1,10-phenanthroline as an activator at pH = 5. The reaction is followed spectrophotometrically by measuring the change in absorbance (A) at 544 nm using a fixed time method (6 min). The reaction variables were optimized in order to achieve the highest sensitivity. The 3s criterion detection limit was 0.7 ng mL^–1, and the relative standard deviation for ten replicate measurements of 16 ng mL^–1 of Ag(I) was 0.95% (n = 10). The method was successfully applied to the determination of silver in expired black and white photographic films and results showed good agreements with those obtained by atomic absorption spectrometry.     

Use of High-Performance Liquid Chromatography–UV and Gas Chromatography–Mass Spectrometry for Determination of the Imidacloprid Content of Honeybees, Pollen, Paper Filters, Grass, and Flowers

Imidacloprid is a new insecticide with a wide range of action. Because honeybees are very sensitive to this substance, two techniques (HPLC–UV and GC–MS) which enable its detection in several matrices of both animal and vegetable origin were used to monitor its possible presence in cultivated land. In the first method quantification of imidacloprid in honeybees was achieved by use of the external standard method; the detection limit was 50 mg kg^–1, the linear range 0.05–1 mg mL^–1, recovery 60–83%, and the imprecision (coefficient of variation) 8.6% for repeatability and 11.8% for reproducibility. Recovery from pollen was 71–98% in the range 0.05–0.5 mg kg^–1. The repeatability was 9.2–13.9%. Imidacloprid can often be found in the environment, not as a simple molecule but as a group of degradation products. The GC–MS method could be used to quantify all these species as oxidation products and to determine the initial quantity of imidacloprid by use of a conversion factor. The liquid chromatographic analysis could be used to detect, in a standard solution, 10 ng mL^–1 derivatized 6-chloronicotinic acid. The linearity was good (R=0.999) over a wide concentration range (10 g mL^–1–10 ng mL^–1). Several samples with different matrices (filter paper placed on an pneumatic corn seed drill, grass, flowers, honeybees, etc.) obtained during the sowing period for imidacloprid-treated corn were analyzed. The quantification limit (LOQ) was 0.005 mg kg^–1 for grass and flowers, 0.002 mg kg^–1 for honeybees, and 0.024 mg kg^–1 for paper filters.     

Microdialysis sampling and high-performance liquid chromatography with chemiluminescence detection for in-vivo on-line determination and study of the pharmacokinetics of levodopa in blood

A simple, reliable, and reproducible method for in-vivo on-line separation and determination of levodopa has been based on microdialysis then high-performance liquid chromatography with chemiluminescence detection. The perfusate is perfused at a flow rate of 5 L min^–1. The concentration of levodopa in the dialysate is determined on line with a chemiluminescence system. The dialysate sample volume is approximately 20 L. The response of the system is linearly related to the concentration of levodopa in the range 1×10^–8 to 1×10^–6 g mL^–1 (r²=0.9995) with a detection limit (3) of 3×10^–9 g mL^–1 and sample throughput of 12 h^–1; RSD is 2.8% (n=11). The method has been successfully used to study the pharmacokinetics of levodopa in vivo; the values of the pharmacokinetics parameters C_max, AUC_0–t and T_max were 16.60, 20.92 ng mL^–1, and 90 min, respectively.     

Determination of Tandospirone and its Impurities in Drug Formulations by LC-UV and LC-MS

This work describes the determination of tandospirone in bulk drug substance and formulated products by a reversed-phase liquid chromatographic method with UV detection. Chromatographic separation was performed on a C₁₈ column with a mobile phase of a binary mixture of methanol and water (70:30, v/v) delivered at a flow rate of 0.5 mL min^–1 and detection was performed at 243 nm. The proposed LC method is selective, precise and accurate for the determination of tandospirone in the presence of its manufacturing impurities with a limit of quantitation of 0.54 g mL^–1. A preliminary study for the identification of the major manufacturing impurities was made by liquid chromatography-mass spectrometry with electrospray ionization source operated in a positive ion mode.     

High-Performance Liquid Chromatographic Assay of Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in Human Plasma using a Solid-Phase Extraction Technique

A selective and sensitive liquid chromatographic method was developed for the determination of zonisamide in small volumes of plasma. Zonisamide and the internal standard methyl 4-hydroxybenzoate were extracted from 0.2 mL of plasma with solid-phase extraction columns and eluted with methanol. Analysis of the extracts was performed on a Symmetry C₁₈ column with ultra-violet spectrophotometric detection. The calibration curve was linear over the concentration range of 0.05–5 g mL^–1 in plasma. Recoveries were reasonable for routine analyses; the limit of quantification was 0.05 g mL^–1 with a signal-to-noise ratio of 5. This method could be useful for the pharmacokinetic study of zonisamide in a limited volume of human plasma and for therapeutic drug monitoring.     

Ultraviolet Spectrophotometric Determination of Primary Amine-Containing Drugs via Their Charge-Transfer Complexes with Tetracyanoethylene

A spectrophotometric method is described for the quantitative determination of some drugs containing a primary amino-group, i.e. amantadine hydrochloride, tranylcypromine sulfate and tranexamic acid in their pure forms as well as in some pharmaceutical dosage forms. The procedure is based on the formation of a charge-transfer complex between tetracyanoethylene (TCNE) as -acceptor and the studied drugs as n-donors in the presence of acetonitrile as solvent. The spectra of the complexes show maxima at 330nm with high apparent molar absorptivities (from 2.1 × 10³ to 2.2 × 10⁴ L·mol^–1 cm^–1). Beers law is obeyed in the concentration ranges of 25–75 µg mL^–1, 2–30µgmL^–1 and 5–25µg mL^–1, and the mean percent recoveries are 99.68±0.92, 100.3±0.75 and 99.8±0.76 for amantadine hydrochloride, tranylcypromine sulfate and tranexamic acid, respectively. The proposed method is simple and can be applied to determine these drugs in their pharmaceutical dosage forms. The results compare favorably with those of reported methods.     

Liquid Chromatographic Determination of Four Purine Bases Using Porous Graphitic Carbon Column

A high performance liquid chromatographic method has been developed for the simultaneous determination of hypoxanthine (Hx), xanthine (Xa), guanine (Gu) and adenine (Ad) in shellfish. The separation of these compounds was performed on a porous graphitic carbon column (100 × 4.6 mm I.D.) using acetonitrile-phosphate buffer (23/77 v/v) containing 0.5% trichloroacetic acid at pH 2.0 as the mobile phase. The flow rate was fixed to 1 mLmin^–1 and the detection was monitored at 254 nm. Calibration curves were found to be linear in the concentration range from 0.3 to 100 gmL^–1 with correlation coefficients (r²) > 0.999. The lowest detectable concentrations of Hx, Xa, Gu and Ad were 0.04, 0.06, 0.08, 0.07 g mL^–1 respectively. Recovery of all purine standards added to sea urchin gonad was above 97%. The proposed method was successfully applied for the determination of Hx, Xa, Gu and Ad in sea urchin gonad extract.     

Trace element determination in seawater by ICP-SFMS coupled with a microflow nebulization/desolvation system

Direct and simultaneous determination of Al, Ag, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Pb, Sb, U, V and Zn in diluted (1:10 v:v) seawater from the Antarctic Ocean and the Venice Lagoon at the ng mL^–1 and pg mL^–1 level has been performed by using an inductively coupled plasma sector field mass spectrometer (ICP-SFMS). Samples were analysed by using a PFA microflow nebulizer coupled with a desolvation system or a PFA microflow nebulizer coupled with a Teflon spray chamber, respectively. Measurements were carried out at low (LR, m/m=300), medium (MR, m/m=3,000) and high (HR, m/m=7,500) resolutions depending on the studied isotope. To avoid contamination, sample pre-treatment was carried out in a clean laboratory equipped with a Class 100 vertical laminar flow hood. Concentration ranges (minimum–maximum in ng mL^–1) found in the Antarctic seawater samples (in depth profiles) were: Ag 0.0004–0.0018, As 0.69–1.32, Cd 0.031–0.096, Co 0.018–0.065, Cr 0.18–0.46, Cu 0.04–1.58, Fe 0.13–1.63, Mn 0.02–0.12, Mo 5.97–12.46, Pb 0.007–0.074, Sb 0.033–0.088, U 0.5–1.9, V 0.6–2.5 and Zn 0.16–0.80. Concentration ranges (min–max in ng mL^–1) found in the Venice Lagoon water samples (temporal profile from a benthic chamber experiment) were: Al 0.24–0.61, Ag 0.007–0.031, As 1.42–2.27, Cd 0.050–0.182, Co 0.440–1.461, Cr 0.15–0.34, Cu 0.81–2.46, Fe 0.25–1.66, Mn 11.6–31.7, Mo 6.50–10.6, Pb 0.047–0.225, Sb 0.240–0.492, U 1.7–3.3, V 1.3–2.8 and Zn 5.20–21.5. The detection limits range between 0.06 pg mL^–1 for Ag and U to 15 pg mL^–1 for Fe. In order to check the accuracy of the analytical procedure, measurements of the trace elements in a certified reference material (coastal Atlantic seawater, CASS-4-NRCC) were compared with the certified values. In addition, the results from the Antarctic and Venice Lagoon samples were compared with those obtained by using different analytical techniques.