Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

Simultaneous RP-LC Determination of Artesunate and Amodiaquine in Pharmaceutical Preparations

A simple, fast and precise reversed phase liquid chromatographic method was developed for the simultaneous determination of artesunate (AS) and amodiaquine (AD) in combined pharmaceutical dosage form. Chromatographic separation of the two drugs was performed on a BDS Hypersil C18, 100 mm × 4.6 mm, 3 μm particle size column as stationary phase with a mobile phase comprising of phosphate buffer (pH 3.0 with orthophosphoric acid) and acetonitrile in the proportion of 50:40 (v/v), at a flow rate of 0.8 mL min^?1 and UV detection at wavelength 210 nm for AS and 300 nm for AD using photo diode array detection. The proposed method was validated for specificity, accuracy, linearity, range, precision and was successfully applied to the simultaneous determination of AS and AD in the combined fixed dosage form without any excipient’s interference.     

Validation and Application by LC for Simultaneous Determination of Imperatorin and Isoimperatorin in Traditional Chinese Medicinal Preparations Containing Radix Angelicae dahuricae

A reversed-phase liquid chromatographic method was established for the simultaneous determination of imperatorin and isoimperatorin in eleven kinds of traditional Chinese medicinal preparations (TCMPs) containing Radix Angelicae dahuricae (Chinese herbal name: Baizhi). Imperatorin and isoimperatorin were successfully separated on an Ultimate XB-C₁₈ column (150 mm × 4.6 mm i.d., 5 μm). The mobile phase was a mixture of acetonitrile and 50 mmol L^?1 sodium acetate (pH 3.5) buffer (45:55, v/v), employing isocratic elution at a flow rate of 1.0 mL min^?1. Detection was accomplished at 302 nm. Regression equations revealed good linear relationship between the peak areas of the constituents and their concentrations (correlation coefficients: 0.9995 for imperatorin, 0.9991 for isoimperatorin). The recoveries were between 97.38% and 103.78%. The proposed method has been successfully applied to the simultaneous determination of imperatorin and isoimperatorin in eleven kinds of TCMPs containing Baizhi.     

Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets

In the present study, a simple, rapid and precise liquid chromatographic method was developed and validated for the simultaneous determination of glimepiride and metformin in sustained release formulation. The separation was achieved on a Nucleosil 100-5SA column, 250 × 4.6 mm i.d., 5 μm using a mobile phase composed of 1.7 % ammonium dihydrogen phosphate buffer pH 3.0: acetonitrile (70:30 v/v).The instrumental settings were a flow rate of 1.0 ml min^?1 and a detector wavelength of 230 nm. The retention time of glimepiride and metformin were 5.1 and 11.3, respectively. The developed method was validated in terms of specificity, sensitivity, linearity, range, accuracy, precision and ruggedness. The proposed method was successfully applied to the sustained release pharmaceutical dosage form for the simultaneous determination of glimepiride and metformin without any interference by excipients.     

Simultaneous Determination of Nickel and Cobalt as Chelates with 1-(2-Pyridylazo)-2-naphthol Using an LC Switching Column Method

A simple liquid chromatographic method was developed for the separation and simultaneous determination of cobalt and nickel as chelates with 1-(2-pyridylazo)-2-naphthol (PAN). The method, using a switching column technique for the on-line purification and separation, enables to reach the sub-microgram per litre concentration level excluding off-line sample treatment with the exception of the derivatization reaction. Two small-sized columns packed with CN- and C₄-bonded stationary phases were selected and used considering their complementary behaviour with respect to chelated Co and Ni ions. The analysis was performed within 10 min using an optimised eluent (water–acetonitrile–methanol–tetrahydrofuran, 40:45:10:5, v/v/v/v) containing Tween 40 (10^?3 M) and acetate buffer (5 × 10^?3 M, pH 4.8). Detection was performed by UV-vis spectrophotometry (λ = 565 nm) permitting to reach quantification limits of 0.9 and 0.5 μg L^?1 for Co and Ni, respectively.     

An Improved HPLC Method for Simultaneous Analysis of Losartan Potassium and Hydrochlorothiazide with the Aid of a Chemometric Protocol

An experimental design method was used for fast, simple, and accurate high-performance-pressure liquid chromatograpy (HPLC) determination of losartan and hydrochlorothiazide in combined dosage forms. This method avoids the disadvantages of the traditional analytical approach, which is time-consuming, involves a large number of runs, and does not allow the determination of multiple interacting parameters. On the basis of preliminary experiments, three independent variables (methanol content, pH value of the mobile phase, and flow rate) were selected as input, and as dependent variables, five responses (retention time of hydrochlorothiazide, retention time of losartan, asymmetry of hydrochlorothiazide peak, asymmetry of losartan peak, and resolution) were chosen. A full 2³ factorial design was used to determine which factors had an effect on the studied response. Afterwards, experimental design was used to optimize these influencing parameters in the previously selected experimental domain. After optimizing the experimental conditions, a separation was conducted on a Zorbax C₈ (150 mm × 4.6 mm, 5 μm particle size) column with a mobile phase consisting of methanol–acetonitrile–acetate buffer 45:20:35 v/v/v, pH 4.8 with flow rate of 0.82 mL min^?1 and column temperature of 25 °C. The developed method was successfully applied to simultaneous separation of these active drug compounds in their commercial pharmaceutical dosage forms.     

Simultaneous Determination of Etodolac and Acetaminophen in Tablet Dosage Form by RP-LC

A simple, specific, precise and accurate reverse phase liquid chromatographic (RP-LC) method has been developed for the simultaneous determination of etodolac and acetaminophen in tablet dosage form. The chromatographic separation was achieved on a BDS Hypersil C18, 100 mm × 4.6 mm, 5 μm column at a detector wavelength of 274 nm using an isocratic mobile phase consisting of a mixture of 0.05% aqueous orthophosphoric acid and acetonitrile in the ratio of 50:50 (v/v) at a flow rate of 1.0 mL min^?1. The retention times for etodolac and acetaminophen were found to be 1.32 and 4.24 min, respectively. The method was validated for the parameters like specificity, linearity, precision, accuracy and robustness. The method was found to be specific and stability indicating as no interfering peaks of impurities, degradent and excipients were observed. The square of correlation coefficients (R ²) for etodolac and acetaminophen were 0.9996 and 0.9998 while percentage recoveries were 101.32 and 100.94%, respectively. Intra- and inter-day relative standard deviations for both the components were <2.0%. The proposed RP-LC method can be applied for the routine analysis of commercially available formulations of these drugs either as such or in combination.     

UPLC-ESI-MS-MS Determination of Three β2-Agonists in Pork

The chromatographic behaviour of bupropion hydrochloride, a basic drug of pK _a 7.9, has been investigated under reversed-phase ion-pairing conditions and the results were used to develop a method for analysis of bupropion hydrochloride in pharmaceuticals. Chromatographic separation of bupropion hydrochloride and carbamazepine (used as internal standard) was performed on a C₈ column (150 mm × 4.6 mm i.d., 3.5-μm particle), with 40:10:50 (v/v) methanol–acetonitrile–phosphate buffer (20 mm, pH 3.0), containing 10 mm 1-heptane sulfonic acid sodium salt (1-HSA), as optimum mobile phase at a flow rate of 1.0 mL min^?1. UV detection was at 254 nm. The fully validated method enables reproducible and selective analysis of bupropion hydrochloride in pharmaceuticals.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.     

An LC Method for Quantification of 2,3-Dichlorobenzoyl Cyanide and Its Related Regio Isomers

A simple, sensitive and selective gradient reversed phase LC method has been developed for separation and determination of 2,3-dichlorobenzoyl cyanide and its regio isomers which is an intermediate of lamotrigine pure form. The separation was achieved on a reversed phase C-18 column using 0.01 M ammonium acetate buffer at pH 5.5 and methanol (50:50 v/v) mixture as solution A and methanol, water mixture (90:10 v/v) as solution B in a gradient elution at a flow rate of 1.5 mL min^?1 with UV detection at 215 nm. The method is found to be selective, precise, linear, accurate and also robust. It was not only used for quality assurance, but also monitoring the synthetic reactions involved in the process development of Lamotrigine. The LC method is found to be simple, rapid, specific and reliable for the determination of unreacted levels of raw materials and isomers in reaction mixtures and finished product Lamotrigine. The method was fully validated as per ICH guidelines and results from validation confirm that the method is highly suitable for its intended purpose.     

Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min^?1. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL^?1 (r = 0.9999) for TR and 40–160 μg mL^?1 (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.     

Estimation of Perospirone in Human Plasma by LC–MS–MS and Its Application to Pharmacokinetics Study

A sensitive liquid chromatography-tandem-mass spectrometry method was developed and validated for the determination of perospirone in human plasma, using quetiapine as internal standard. Plasma samples were extracted from 1 mL of plasma using n-hexane. Chromatographic separation was performed on an Agilent Zorbax SB C₁₈ column with a mobile phase of 5 mM ammonium acetate solution-methanol (12:88, v/v, adjusted to pH 3.8 with glacial acetic acid) at a flow rate of 0.2 mL min^?1. The chromatographic separation was achieved in less than 4.6 min. The linearity was established over the concentration range of 0.05–20 ng mL^?1. Both of the intra- and inter-batch standard deviation was less than 9.8%. The method was successfully applied to study the pharmacokinetic parameters of perospirone hydrochloride tablets in healthy Chinese volunteers.     

Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation

Two methods are described for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminium sheets of silica gel 60 F₂₅₄ using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil-100 ODS-3–C₁₈ column from MZ-Analysetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (pH = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation.     

LC Determination of Podophyllum Lignans and Flavonoids in Podophyllum emodi Wall.var.chinesis Sprague

A high-performance liquid chromatographic method for the simultaneous separation and determination of nine podophyllum lignans and two flavonoids in Podophyllum emodi Wall.var.chinesis Sprague was established. The separation was performed with the following condition: column, Kromasil-C18, 5 μm, 15 × 0.46 cm; solvent A, 25 mM phosphate buffer, pH 2.5; solvent B, methanol; gradient, 50/50/70% B at 0/13/33 min; flow rate, 0.8 mL min^?1; detection, UV 270 nm. β-Apopicropodophyllin was used as the internal standard. The samples of P. emodi Wall.var.chinesis Sprague from different sources were examined by the developed method. In addition, three methods for sample preparation were discussed. It is shown from the results that refluxing in chloroform displays high extraction efficiency.     

Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets

In the present study, a simple, rapid and precise liquid chromatographic method was developed and validated for the simultaneous determination of glimepiride and metformin in sustained release formulation. The separation was achieved on a Nucleosil 100-5SA column, 250 × 4.6 mm i.d., 5 μm using a mobile phase composed of 1.7 % ammonium dihydrogen phosphate buffer pH 3.0: acetonitrile (70:30 v/v).The instrumental settings were a flow rate of 1.0 ml min⁻¹ and a detector wavelength of 230 nm. The retention time of glimepiride and metformin were 5.1 and 11.3, respectively. The developed method was validated in terms of specificity, sensitivity, linearity, range, accuracy, precision and ruggedness. The proposed method was successfully applied to the sustained release pharmaceutical dosage form for the simultaneous determination of glimepiride and metformin without any interference by excipients.

     

Simultaneous Quantitation of Tetrahydropalmatine and Protopine in Rabbit Plasma by HPLC–PAD, and Application to Pharmacokinetic Studies

A reversed-phase high-performance liquid chromatographic method coupled with photodiode-array detection (HPLC–PAD) has been developed and validated for simultaneous quantitation of dl-tetrahydropalmatine (dl-THP) and protopine (PTP), two active constituents of the herbal medicine Rhizoma Corydalis, in rabbit plasma. The pharmacokinetics of dl-THP and PTP were researched in rabbits after oral administration of extracts of Rhizoma Corydalis. Optimum separation was achieved on a reversed-phase column with methanol–phosphate buffer (pH 7.75, 65:35, v/v) as mobile phase. The experimental results showed the intra-day and inter-day precision and accuracy of the method were satisfactory for simultaneous determination of dl-THP and PTP at low, middle, and high concentrations. The lower limits of quantitation were 2.05 ng mL^?1 for dl-THP and 2.10 ng mL^?1 for PTP. Plasma concentration–time data for dl-THP were best fitted by the two-compartment linear pharmacokinetic model whereas data for PTP were best fitted by the one-compartment model.     

Development of Validated Chromatographic Methods for the Simultaneous Determination of Metronidazole and Spiramycin in Tablets

Two chromatographic methods have been described for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in their mixtures. The first method was based on a high performance thin layer chromatographic (HPTLC) separation of the two drugs followed by densitometric measurements of their spots at 240 nm. The separation was carried out on Merck TLC aluminum sheets of silica gel 60 F₂₅₄ using methanol: chloroform (9:1, v/v) as a mobile phase. Analysis data was used for the linear regression line in the range of 1.0–2.0 and 0.8–2.0 μg band⁻¹ for MET and SPY, respectively. The second method was based on a reversed-phase liquid chromatographic separation of the cited drugs on a C-18 column (5 μm, 250 × 4.6 mm, i.d.). The mobile phase consisted of a mixture of phosphate buffer of pH 2.4 and acetonitrile (70:30, v/v). The separation was carried out at ambient temperature with a flow rate of 1.0 mL min⁻¹. Quantitation was achieved with UV detection at 232 nm based on peak area with linear calibration curves at concentration ranges 0.4–50.0 and 0.5–50.0 μg mL⁻¹ for MET and SPY, respectively. The proposed chromatographic methods were successfully applied to the determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with ICH guidelines; in terms of linearity, accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.

     

Determination of Carvedilol and its Impurities in Pharmaceuticals

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for separation of carvedilol and its impurities from Karvileks tablets. The best separation was achieved on a 100 mm × 4.6 mm, 5 µm particle size, Chromolit RP 8e column. Use of acetonitrile-water, 45:55 (v/v), adjusted to pH 2.5 with formic acid, as mobile phase at a flow rate of 0.5 mL min^?1 enabled acceptable resolution of carvedilol, in large excess, from possible impurities, in a short elution time. UV detection was performed at 280 nm. Linearity, accuracy, precision, selectivity, and robustness were validated and found to be satisfactory. Overall, the proposed method was found to be highly sensitive, suitable, and accurate for quantitative determination of carvedilol and its impurities in dosage forms and in raw materials.     

Simultaneous Determination of Carbamazepine and its Active Metabolite Carbamazepine-10,11-epoxide in Human Plasma by UPLC

A simple method for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide by ultra performance liquid chromatography (UPLC) with ultraviolet absorbance detection (TUV) was developed. The method involves a two-step protein precipitation by liquid–liquid extraction. Phenytoin sodium was used as the internal standard. The separation was carried out on Acquity C18 column with acetonitrile:methanol:KH₂PO₄ buffer (adjusting pH to 4.6 with 85% o-phosphoric acid) (180/180/170, v/v/v) as the mobile phase at a flow rate of 0.4 mL min⁻¹. Linear detection response was obtained for concentrations ranging from 50 to 5,000 ng mL⁻¹. The limit of quantification (LOQ) was 50 ng mL⁻¹. The method was validated successfully for the determination of carbamazepine and its active metabolite carbamazepine-10,11-epoxide, which can be applied through pharmacokinetics and bioequivalence studies.