Development and Validation of Spectrophotometric Methods for the Determination of Cefetamet in Pharmaceutical Dosage Forms

Two simple and sensitive spectrophotometric methods for the determination of cefetamet in either pure form or in its pharmaceutical formulations were described. The method Ⅰ is based on the interaction of 3-methylbenzo[d]- thiazolin-2-one hydrazone （MBTH） with cefetamet in the presence of freshly prepared ferric chloride in a neutral medium. The resulting blue colored product has λmax at 628 nm. The method Ⅱ describes the reduction of ferric ion by the drug to ferrous ion followed by a complex formation reaction with 1,10-phenanthroline （1,10-phen） to form an orange red colored chromogen exhibiting 2max at 510 nm. The products are stable for more than 5 and 8 h respectively. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed methods. Both methods are highly reproducible and have been applied to a wide variety of pharmaceutical preparations and the results are comparable with those of official methods.     

Development and Validation of an HPTLC–Densitometric Method for Determination of ACE Inhibitors

A high-performance thin layer chromatographic method coupled with densitometric analysis has been developed for measurement of benazepril and cilazapril, both pure and in their commercial dosage forms. The active substances were extracted from tablets with methanol (mean recovery 102%) and chromatographed on silica gel 60 F₂₅₄ HPTLC plates in horizontal chambers with ethyl acetate–acetone–acetic acid–water, 8:2:0.5:0.5 (v/v), as mobile phase. Chromatographic separation of these ACE inhibitors was followed by UV densitometric quantitation at 215 nm. Calibration plots were constructed in the range 0.4 to 2.0 g L^–1 for benazepril (2.0–10.0 g spot^–1) and from 0.5 to 1.5 g L^–1 for cilazapril (4.0–12.0 g spot^–1) with good correlation coefficients (r 0.990). The method was used to determine benazepril and cilazapril in pharmaceutical preparations with satisfactory precision (1.4% < RSD < 5.6%) and accuracy (1.7 < RE < 5.1).     

LC–Tandem-MS Development and Validation for the Determination of Tegaserod in Beagle Dog Plasma and Its Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography-tandem mass spectrometry method for quantitative determination of tegaserod was developed and validated over the linearity range 1.0–200.0 ng mL^?1 with 0.5 mL of plasma using diphenhydramine as an internal standard. Liquid–liquid extraction using ethyl ether was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the atmospheric pressure chemical ionization technique. The mobile phase consisted of methanol–water–formic acid (80:20:1, v/v/v), at a flow rate of 0.6 mL min^?1. In the positive mode, tegaserod produced a protonated precursor ion at m/z 302 and a corresponding product ion at m/z 173. The internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The intra- and inter-day accuracy at all levels fell in the ranges of 100.72–102.75% and 100.61–105.45%, and the intra- and inter-day precision were in the ranges of 4.20–5.74% and 1.90–4.17%, respectively. The method was successfully applied to a pharmacokinetic study of tegaserod after an oral administration of two kinds of tegaserod preparations to beagle dogs.     

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

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

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.     

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.     

Development of an RPLC-based Method for Measurement of Indoprofen Stability and Validation of the Method

A method for quantitative measurement of the photochemical decomposition of the anti-inflammatory agent, indoprofen （INP） is descriped. An RPLC-based assay that could determine the extent of degradation of INP in a rapid, sensitive, and accurate manner was developed. The method was validated under photoirradiation. Quantitation was monitored with an Inertsil ODS-3V column using a mobile phase of acetonitril and 1% HOAc solution in deionized H2O. Statistics relevant to the system criteria, peak integrity and resolution among the parent drug and its degradation products were performed. From the intra- and inter-day tests, the coefficients of variation were found to range from 0.59% to 4.25% for the former and from 0.71% to 4.86% for the latter. The good selectivity and specificity of this RPLC-based procedure render it suitable for measurements of INP stability.     

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.     

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

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

     

LC–MS Method for the Sensitive Determination of Metoclopramide: Application to Rabbit Plasma, Gel Formulations and Pharmaceuticals

To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C₁₈ column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min^?1. The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL^?1 with a limit of detection of 0.077 pg μL^?1 for standard solutions and 2.5–200 pg μL^?1 with a limit of detection of 0.42 pg μL^?1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H₂O₂, HCl and NaOH were also investigated.     

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.     

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.

     

Stability-Indicating Liquid Chromatography–Spectrophotometric UV Method for the Simultaneous Determination of Marbofloxacin,Dexamethasone and Clotrimazole in a Liquid Pharmaceutical Dosage Form

A novel, simple and reliable reversed-phase liquid chromatography (LC)–spectrophotometric UV stability-indicating method was developed and validated for the simultaneous assay of marbofloxacin, clotrimazole and dexamethasone acetate in the presence of their impurities and degradation products in a pharmaceutical formulation for veterinary use. A C18 (75 × 4.6 mm, 4 µm) column was used with an acetonitrile–ammonium acetate mixture as mobile phase delivered with gradient elution. A diode-array detection was used in the 200–400 nm range and the detection wavelength was set at 260 nm. Validation carried out on the pharmaceutical dosage form, according to Veterinary International Conference on Harmonization guidelines, demonstrated excellent specificity, linearity, precision, accuracy and robustness. Excellent specificity with respect to vehicle and degradation products obtained after forced degradation (i.e., oxidation, acid, alkaline and thermal degradation) was demonstrated. As for linearity, the LC–UV assay method is applicable in the 0.180–0.420 mg mL^?1 concentration range for marbofloxacin (r ² = 0.99), 0.060–0.140 mg mL^?1 for dexamethasone acetate (r ² = 0.97) and 0.600–1.400 mg mL^?1 for clotrimazole (r ² = 0.98). Very good repeatability (RSD < 0.8 %) and inter-day precision (RSD < 2.5 %) were observed for all analytes. Accuracy was in the 93–104 %, 98–111 % and 99–108 % confidence interval (95 %) for marbofloxacin, dexamethasone acetate and clotrimazole, respectively. The variations (±20 %) of mobile phase flow rate and pH, and oven column temperature did not exhibit an impact on the analyte content accuracy, demonstrating the robustness of the method. The LC–UV method here developed and validated may be used routinely for quality control.     

Stability-Indicating Liquid Chromatography–Spectrophotometric UV Method for the Simultaneous Determination of Marbofloxacin,Dexamethasone and Clotrimazole in a Liquid Pharmaceutical Dosage Form

A novel, simple and reliable reversed-phase liquid chromatography (LC)–spectrophotometric UV stability-indicating method was developed and validated for the simultaneous assay of marbofloxacin, clotrimazole and dexamethasone acetate in the presence of their impurities and degradation products in a pharmaceutical formulation for veterinary use. A C18 (75 × 4.6 mm, 4 µm) column was used with an acetonitrile–ammonium acetate mixture as mobile phase delivered with gradient elution. A diode-array detection was used in the 200–400 nm range and the detection wavelength was set at 260 nm. Validation carried out on the pharmaceutical dosage form, according to Veterinary International Conference on Harmonization guidelines, demonstrated excellent specificity, linearity, precision, accuracy and robustness. Excellent specificity with respect to vehicle and degradation products obtained after forced degradation (i.e., oxidation, acid, alkaline and thermal degradation) was demonstrated. As for linearity, the LC–UV assay method is applicable in the 0.180–0.420 mg mL⁻¹ concentration range for marbofloxacin (r ² = 0.99), 0.060–0.140 mg mL⁻¹ for dexamethasone acetate (r ² = 0.97) and 0.600–1.400 mg mL⁻¹ for clotrimazole (r ² = 0.98). Very good repeatability (RSD < 0.8 %) and inter-day precision (RSD < 2.5 %) were observed for all analytes. Accuracy was in the 93–104 %, 98–111 % and 99–108 % confidence interval (95 %) for marbofloxacin, dexamethasone acetate and clotrimazole, respectively. The variations (±20 %) of mobile phase flow rate and pH, and oven column temperature did not exhibit an impact on the analyte content accuracy, demonstrating the robustness of the method. The LC–UV method here developed and validated may be used routinely for quality control.

     

RP‐HPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma

Abstract

A simple reverse‐phase high‐performance liquid chromatographic method for the determination of tenofovir disoproxil fumarate (TDF) in pharmaceutical formulations and human plasma samples has been developed and validated. Piroxicam (PRX) was used as an internal standard. The assay of the drug was performed on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) with UV detection at 259 nm. The mobile phase consisted of acetonitrile–water mixture in the ratio of 75∶25, and a flow rate of 1 ml/min was maintained. The standard curve was linear over the range of 0.2–10 µg/ml (r ²=0.9966). Analytic parameters have been evaluated. Within‐day and between‐day precision as expressed by relative standard deviation was found to be less than 2%. The method has been applied successfully for the determination of TDF in spiked human plasma samples and pharmaceutical formulations.     

Development and Validation of a GC–MS Method for the Determination of Methyl and Ethyl Camphorsulfonates in Esomeprazole Magnesium

A rapid gas chromatography–mass spectrometry method for the identification and determination of two carcinogenic impurities viz. methyl camphorsulfonate (MCS) and ethyl camphorsulfonate (ECS) in esomeprazole magnesium (EOM) is developed for the first time. The factors affecting method development and the fragmentation patterns of MCS and ECS based on the mass spectral analysis are described. The limit of detection and limit of quantitation values for both compounds were established as 3 and 10 ppm, respectively with respect to 50 mg mL^?1 of EOM. The method is linear within the range of 10–120 ppm and found to be precise, accurate, specific and robust.