Chromatographic Method For Simultaneous Quantification Research Articles

A validated liquid chromatographic method for simultaneous quantification of Lidocaine hydrochloride and Diltiazem hydrochloride

A validated liquid chromatographic method for simultaneous quantification of Lidocaine hydrochloride and Diltiazem hydrochloride

Validated high‐performance thin layer chromatographic method for simultaneous quantification of rifampicin and quercetin in liquisolid formulation using fractional factorial design in robustness study

AbstractThe present research study focuses on the development of the high‐performance thin‐layer chromatographic method using the design of experiment approach for the simultaneous estimation of Rifampicin and Quercetin. Chromatographic separation was performed on Aluminium plates precoated with silica gel 60 F254 using chloroform:methanol:formic acid:ethyl acetate:benzene (4:1.8:0.3:1.7:1, v/v/v/v/v) as optimized mobile phase. A fractional factorial design was applied for the robustness study and the independent variables selected were mobile phase composition (A), solvent front (B), chamber saturation time (C), and wavelength (D). It was statistically revealed that the volume of ethyl acetate affected the Rf of both drugs resulting in stricter control of ethyl acetate volume compared to the other three variables. Rf for Rifampicin and Quercetin was 0.35 and 0.71 at 347 nm. The linear concentration range was 100–600 ng/band for both drugs. The %recovery ranged between 95.25% and 96.71% for Rifampicin and 95.33%–96.86% for Quercetin. The method was validated by determination of linearity, precision, accuracy, and specificity according to Q2(R1) guidelines, and the %relative standard deviation values were less than 2% for both drugs. A simple, accurate, and reproducible high‐performance thin‐layer chromatographic method was developed, for routine quality control testing of pharmaceutical formulation.

An Effective Chromatographic Method for Simultaneous Quantification of Antidiabetic Drugs Alogliptin Benzoate and Pioglitazone HCl in Their Tablet Dosage Form: Implementation to In vitro Dissolution Studies and Uniformity of Dosage Unit.

In patients with Type 2 diabetes, a combination of Alogliptin and Pioglitazone medications, together with diet and physical activity, are used to improve glycemic control. Eco-friendly, cost-effective, and precise stability-indicating RP-HPLC method was developed and validated for the identification and quantification of Alogliptin and Pioglitazone in their tablet dosage form, as well as implementation to in vitro dissolution studies and uniformity of dosage unit. Isocratic separation is conducted at ambient temperature on the InertSustain C18 Analytical Column (150 × 4.6mm, 5μm) using mobile phase comprising 50mM of ammonium dihydrogen phosphate and 5.0mM of heptane sulfonic acid:acetonitrile (45:55, v/v) at a flow rate of 1.3mL/minute. Calibration curves are conducted in the linearity range of 1-40μg/mL of Alogliptin and 2.5-75μg/mL of Pioglitazone with a correlation value >0.9995 and satisfactory recovery findings between 99 and 100%. The degraded samples are analyzed under relevant stress conditions as acidity, alkalinity, thermal and oxidation. The active components in finished products were subjected to a content uniformity test, which showed that they achieved the declared claim's acceptance standards (85-115%). Comparative in vitro dissolution studies are performed for generic products Inhibazone 12.5/30mg FCT and Inhibazone 25/15mg FCT against innovator products Oseni 12.5/30mg FCT and Oseni 25/15mg FCT at suitable FDA dissolution medium and different USP dissolution media and the results are similar. The metrics of the designed method were assessed according to ICH requirements, and all metrics, such as system suitability, linearity, recovery, robustness, LOD, LOQ, specificity and precision, were found to be within required tolerances and no overlapping was found for degradation peaks. Thence, the method can be used in quality control for the analysis of raw material, bulk, finish and stability.

Development and application of a chromatographic method for simultaneous quantification of 5-fluorouracil and imiquimod in drug-in-adhesive topical patches

Topical patches containing 5-fluorouracil and imiquimod would significantly improve the shortcomings of the marketed semi-solid formulations for the treatment of non-melanoma skin cancer. As an analytical method is a fundamental part of formulation development, in this study, a simple, accurate, sensitive and greener analytical method was developed and validated for the simultaneous quantification of 5-fluorouracil and imiquimod. The optimal chromatographic conditions were obtained using a C18 column (250 × 4.6 mm, 5 μm), mobile phase containing 0.1 M sodium acetate buffer (pH 4) and acetonitrile (60:40, v/v), a flow rate of 1 mL/min and injection volume of 10 μL at 25 °C. Signals were detected at 244 nm and 265 nm for imiquimod and 5-fluorouracil, respectively. Validation procedures were performed according to the United States Food and Drug Administration guidelines and the linearity range was found to be 0.78 – 50 μg/mL for both 5-fluorouracil and imiquimod. Other parameters including accuracy, precision, robustness, selectivity and stability were within the acceptable limits. The proposed method was successfully applied to determine the 5-fluorouracil and imiquimod contents simultaneously and their release profiles of a drug-in-adhesive patch. Furthermore, the greenness of the proposed analytical method was comprehensively determined using various greenness evaluation tools.

SIMULTANEOUS METHOD DEVELOPMENT AND VALIDATION OF CHOLINE SALICYLATE AND TANNIC ACID USING RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: The current investigation was pointed at completely unique and reliable high performance liquid chromatographic method for simultaneous quantification of Choline salicylate and Tannic acid. Methods: Chromatographic separation was achieved on a symmetry C18 column (150x4.6mm, 3.5 µ) using isocratic elution with a buffer containing 1 ml of OPA in l lt of HPLC marked water and acetonitrile within the percentage of 60:40 as movable phase with a flow rate of 1.0 ml/min at ambient temperature. Analysis was achieved within 15 min over an honest linearity within the concentration range from 4-60 µg/ml of choline salicylate and 2.5-37.5 µg/ml of tannic acid. Stress conditions of degradation in acidic, alkaline, peroxide and thermal was studied. Results: LOD and LOQ were observed as 1.21 µg/ml, 0.758 µg/ml and 4 µg/ml, 2.5 µg/ml of choline salicylate and tannic acid respectively. Precision and recovery study results were found to be within the suitable limit. Conclusion: This developed method showed reliable, precise, accurate results under optimized conditions. The method was validated as reported by ICH guidelines. Hence it was evident that the proposed method was suitable for regular analysis and quality control of pharmaceutical preparations.

Direct injection green chromatographic method for simultaneous quantification of amoxicillin and amikacin in maternity hospital wastewater (Sagar, India)

Amoxicillin (AMO) and amikacin (AMK) are broad-spectrum antibiotics that are most preferably given post-delivery (normal and cesarian) in the maternity hospitals located in Sagar city (Madhya Pradesh), India. Both the antibiotics make their way through sewage/drainage systems into the environment in the form of metabolized and unmetabolized compounds. Growing concern about the contamination of wastewater by antibiotics requires fast, sensitive and eco-friendly techniques. Therefore a simple, rapid and environmental friendly chromatographic method has been developed for simultaneous determination of AMO and AMK in maternity hospital wastewater samples. A micellar liquid chromatographic (MLC) method was developed with a C18 column (250 mm × 4.6 mm), sodium dodecyl sulphate (SDS; 0.15 M), 1-butanol (7%) as a modifier, pH 5 and photo diode detector (PDA) at 270 nm and 256 nm for AMO and AMK respectively. The method was fast with analysis time below 9 min. In the present MLC method, linearities (r > 0.998), limits of quantification in the range of 0.02–0.04 μg/mL, repeatabilities, and intermediate precision below 4.9% were adequate for the quantification of AMO and AMK. The proposed method can be utilized to detect and quantify both the antibiotics in various samples by hospitals, pharmaceutical companies, pollution control board, municipal corporations, etc.

DOMINATED RP-HPLC METHOD FOR QUALITY CONTROL OF TERNARY PHARMACEUTICAL FORMULATION: GUAIFENESIN/LEVOCETIRIZINE/ AMBROXOL HCL AND STABILITY STUDIES

The present investigation was aimed to establish a liquid chromatographic method for simultaneous quantification of guaifenesin, levocetirizine and ambroxol HCl in ternary fixed dosage form. The three drugs were swiftly resolved using ODS C18 reverse phase column with mobile phase consisting of ammonium phosphate buffer, pH 4.5: acetonitrile (60:40, v/v) at a flow rate of 1.0 mL/min and UV detection at 236 nm. The retention time values were 2.231, 2.772 and 6.309 min, respectively, for guaifenesin, levocetirizine and ambroxol HCl. The response was a linear function of analyte over the concentration range of 12.5-75 µg/mL, 0.625-3.750 µg/mL and 3.75-22.5 µg/mL with correlation co-efficient value of 0.999. Maximum recovery (99.12-101.2 %) was obtained in ternary liquid oral formulation (Leoriv plus syrup). Three drugs were well resolved from their degradation products and net degradation was calculated in acid hydrolysis, base hydrolysis, oxidation, thermal and UV conditions. The proposed method enables the simultaneous quantification of guaifenesin, levocetirizine and ambroxol HCl in routine QC laboratories.

Stability indicating liquid chromatographic method for simultaneous quantification of betamethasone valerate and tazarotene in in vitro and ex vivo studies of complex nanoformulation.

Low-potency corticosteroid betamethasone valerate and vitamin-A tazarotene are used in combination for effective treatment of psoriasis. There is no robust high-performance liquid chromatography analytical technique available for simultaneous estimation of betamethasone valerate and tazarotene in conventional and nanocarriers based formulations. A simple, accurate, robust isocratic high-performance liquid chromatography method was developed for simultaneous estimation of betamethasone valerate and tazarotene in topical pharmaceutical formulations. The developed method was validated as per the regulatory guidelines. The validated method was linear over the concentration range of 150-6000ng/mL (r2 >0.999) at 239nm wavelength. Limits of detection and quantification of two analytes were 50 and 150ng/mL, respectively. The %relative standard deviation for intraday and interday precision was less than 2%. The method was also evaluated in the presence of forced degradation conditions. The developed method was successfully applied for in vitro and ex vivo drug release studies of in-house designed nanoformulations.

Development and validation of stability indicating reversed-phase liquid chromatographic method for simultaneous quantification of methotrexate and teriflunomide in nanoparticles and marketed formulation.

Methotrexate (MTX) and teriflunomide (TEF) are the two most effective disease-modifying antirheumatic drugs used as combination therapy for rheumatoid arthritis and no robust high-performance liquid chromatography (HPLC) method is available for their simultaneous estimation to date. Therefore, we have developed and validated an isocratic reversed-phase HPLC method for simultaneous analysis of MTX and TEF spiked in the form of active pharmaceutical ingredients, tablets and nanoformulations. The best separation was achieved on a BDS, C18 , 4.6 × 250 mm, 5 μm analytical column (Thermo Hypersil) with methanol-ethylammonium formate-potassium dihydrogen phosphate buffer (55 mm, pH 3.5; 65:5:30, v/v) as mobile phase at a flow rate of 0.8 mL/min. All the samples were subjected to force degradation studies. Responses of MTX and TEF were found to be a linear function of concentration over the range 1-50 μg/mL (r2 = 0.9976 and 0.9982). The limits of detection and limit of quantification were 7.74 and 25.82 ng/mL and 10.74 and 35.80 ng/mL, respectively. Degradation products produced under the stress studies did not interfere with the detection of MTX and TEF and therefore the developed method can be regarded as stability indicating.

Ultra performance liquid chromatographic method for simultaneous quantification of plerixafor and related substances in an injection formulation

AbstractPlerixafor (PLX) injections are administered to patients with cancers of lymphocytes (non-Hodgkin’s lymphoma) and plasma cells (multiple myeloma). The main objective of the current study was to develop a short reverse phase chromatographic method for the simultaneous quantification of PLX and its impurities, in an injection formulation, to reduce the time required for these quality tests. Furthermore, the present work describes the role of nonalkyl branched nonquaternary ion pair reagent in improving the peak shape and reducing column equilibration time. The separation of PLX and its related substances is pH dependent (optimum pH = 2.50) and was achieved on an octadecylsilyl (C18) column. The method was validated for its intended purpose in accordance with the current regulatory guidelines for validation. The proposed method can be applied for quality control, release, and stability analyses of active pharmaceutical ingredient, PLX, as well as finished products, PLX injections.

High Performance Liquid Chromatographic Method for Simultaneous Quantification of Naloxone and Oxycodone

High Performance Liquid Chromatographic Method for Simultaneous Quantification of Naloxone and Oxycodone

Liquid Chromatographic Method for Simultaneous Quantification of Alprazolam and Mebeverine in Pharmaceutical Dosage Form

A sensitive, rapid, accurate and reproducible Isocratic, RP-LC method was developed for determination of Alprazolam and Mebeverine in pharmaceutical dosage form. A Sunfire C18, 5 μm column with mobile phase containing methanol: buffer (0.02M KH2PO4) (70:30). The flow rate was 1.0 ml/min and effluents were monitored at 225 nm with help of PDA detector. The retention time was 6.04 min. for ALP and 3.53 min. for MEB. The linearity was in the range of 0.05-40 μg/ ml for ALP and 0.2-40 μg/ ml for MEB. The proposed method was validated with respect to linearity, accuracy, precision, specificity and robustness. Due to simplicity, rapidity and accuracy of method, believe that the method will be useful for routine quality control analysis.

New liquid chromatographic method for simultaneous quantification of Atovaquone and Proguanil with its active metabolite Cycloguanil in human plasma

Abstract: Objective: The aim of the present study is to develop HPLC method for simultaneousquantification of atovaquone and proguanil with its active metabolite cycloguanilin human plasma. Methodology: A specific and accurate highperformance liquid chromatographic method has been developed using Phenyl (150×4.6 mm, 5 μm) column maintained at 18 °C. The separation was achieved using a mobile phase composed of phosphate buffer pH 7.2 and methanol (45:55%). The mobile phase was maintained at flow rate of 0.8 mL/min. The analytes were monitored at 254 nm usingultra-violet detector. The plasma samples extractions was carried out using tert-Butyl Methyl Ether: Dichloromethane (80:20% v/v) mixture. Tramadol was used as an internal standard (ISTD). Result: The developed method was validated as per US FDA guidelines and found to be highly specific, precise and accurate. Moreover, atovaquone, proguanil and cycloguanilwere stable in plasma at various stability conditions. Conclusion: The developed method is simple, economic, and can be used for quantification of said drugs human plasma samples. Key words: Atovaquone, Cycloguanil, Proguanil, Human plasma, Bioanalysis, HPLC.

A modified high performance liquid chromatographic method for simultaneous quantification of skatole and indole in porcine plasma

A simplified high-performance liquid chromatography method to measure skatole and indole in porcine plasma without the use of acetonitrile was developed and validated in this study. The mobile phase consisted of water and methanol used in a gradient programme. Fluorescence detection was performed on the supernatant obtained from plasma after protein precipitation with 100% acetone. Limits of quantification were 0.5 ng∙ml-1 for skatole and 1.0 ng∙ml-1 for indole. Accuracy and precision had less than 12% deviation in the linear ranges (0.5–256 ng∙ml-1 and R2 = 0.9999 for skatole, 1.0–256 ng∙ml-1 and R2 = 0.9999 for indole). The correlation between plasma and serum concentrations was strong for skatole (slope = 1.01, R2 = 0.999) and moderate for indole (slope = 0.65, R2 = 0.95). Analysis of skatole in plasma was in good accordance with our previous acetonitrile-based method (slope = 0.91, R2 = 0.988). The proposed method is suited for rapid routine analysis because of its high selectivity, accuracy and precision. Furthermore, it needs only simple sample preparation and the use of methanol instead of acetonitrile in the mobile phase. This method is of practical use to researchers in the field of boar taint.

Validated high performance thin layer chromatographic method for simultaneous quantification of major iridoids in Vitex trifolia and their antioxidant studies

Negundoside ( 1), agnuside ( 2) and 6′- p-hydroxy benzoyl mussaenosidic acid ( 3) are known bioactive metabolites in Vitex trifolia. In the present study a simple precise and reproducible method was developed for simultaneous quantitation of NS ( 1), AS ( 2) and HMA ( 3) and the antioxidant capacity of above markers has also been determined. Marker compounds have been resolved using silica gel 60 F 254 plates, petroleum ether (60–80)/toluene/acetone/water (10:10:80:2 v/v/v/v) as the mobile phases. The method does not employ any derivatisation procedure and can be used as a quality control tool for routine analysis of drugs V. trifolia and V. negundo together with their commercial extracts. NS ( 1), AS ( 2) and HMA ( 3) showed significant activity in DPPH and NO radical scavenging assays.

SIMULTANEOUS DETERMINATION OF GLIMEPIRIDE AND ATORVASTATIN IN HUMAN SERUM BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY: APPLICATION TO PHARMACOKINETIC STUDY

A simple and sensitive high performance liquid chromatographic method for simultaneous quantification of glimepiride (GMP) and atorvastatin (ATN) in human serum was developed and validated. GMP, ATN, and internal standard (IS), pioglitazone (PG) were extracted into dichloromethane and methanol solvent system and separated using an isocratic mobile phase on an Inertsil C18 column. The eluent was monitored by UV detector at 230 nm. The linearity range of proposed method was 1–1600 ng mL−1 for each analyte. The retention times for GMP, ATN, and IS were found to be 9.8, 6.92, and 7.96 min, respectively. The intra-day and inter-day coefficient of variation and percentage error values of the assay method were less than 15% and mean recovery was more than 96, 93, and 95% for GMP, ATN, and IS, respectively, and the method was found to be precise, accurate, and specific. The method was successfully applied for pharmacokinetic study of GMP and ATN after oral administration to patients. The CMax, TMax, and AUC0–12 of GMP and ATN were found to be 355.3 ng mL−1, 3.1 hr, 2167.5 ng h mL−l and 12.6 ng mL−1, 2.6 hr, 80.8 ng h mL−l, respectively.

Ultra-performance liquid chromatographic method for simultaneous quantification of HIV non-nucleoside reverse transcriptase inhibitors and protease inhibitors in human plasma

A fast ultra-performance liquid chromatographic with diode-array detection method has been developed and validated for the determination of six protease inhibitors (indinavir, amprenavir, saquinavir, atazanavir, lopinavir, and ritonavir) and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). After liquid-liquid extraction of 0.5 mL plasma with methyl-tert-butyl ether, the analytes were separated on a ACQUITY UPLC BEH® C18 column (2.1 × 150 mm, p.d. 1.7 μm) column eluted with a gradient of acetonitrile and triethylammonium phosphate buffer 5 mmol L-1 pH 3.0. The total run time was 9.5 min. Calibration curves were linear in the range 0.1 to to 10.0 μg mL-1. The lower limit of quantitation was 0.1 μg mL-1 for all drugs. Accuracy ranged from 94.9 to 103.5%. Both interday and intraday coefficients of variation were less than 7.7% for all analytes. The extraction yields were greater than 88.2%.

Biotransformation of nodakenin and simultaneous quantification of nodakenin and its aglycone in incubated system of human intestinal bacteria by HPLC method

When nodakenin (1) was anaerobically incubated with human intestinal bacteria, nodakenetin (2) was found as a main biotransformed product. We developed a simple and selective reversed-phase high-performance liquid chromatographic method for simultaneous quantification of 1 and 2 in incubated system of human intestinal bacteria with 1. Chromatographic separation of 1 and 2 was performed on an analytical C18 column, with a mobile phase of MeOH–H2O (4:6, v/v) at a flow rate of 1.0 ml/min and the UV detection was at 330 nm. The calibration curves were linear over the range of 0.15–24.0 μg/ml for 1 and 0.7–13.2 μg/ml for 2. The lower limits of detection and quantification were 0.01 and 0.1 μg/ml for 1, and 0.005 and 0.05 μg/ml for 2. The recoveries were (87.66 ± 1.66), (79.89 ± 2.53), and (82.96 ± 5.61)% at 1.0, 2.0, and 8.0 μg/ml, respectively, for 1 and (88.32 ± 4.12), (78.15 ± 4.39), and (76.22 ± 3.29)% at 1.0, 4.0, and 16.0 μg/ml, respectively, for 2. The intra- and interday precision and accuracy were validated by relative standard deviation, which were in the ranges of 1.25–4.16 and 2.16–6.12% for 1, and 1.98–6.45 and 2.56–4.57% for 2, respectively. This method has been applied to the simultaneous quantitation of 1 and 2 in incubated system of human intestinal bacteria with 1.

High‐performance liquid chromatographic method for simultaneous quantification of eight major biologically active ingredients in ‘Da‐Chai‐Hu‐Tang’ Preparation

A simple, sensitive and specific high-performance liquid chromatographic method has been developed for the first time to simultaneously determine the eight major biologically active ingredients, namely paeoniflorin, naringin, sennoside A, baicalin, baicalein, saikosaponin a, rhein and emodin of the Chinese herbal formula Da-Chai-Hu-Tang. The contents of these marker substances in Da-Chai-Hu-Tang extract could be easily determined within 85 min. The assay was reproducible and accurate with overall intra-day variations and accuracy of less than 2% and more than 97.9%, respectively. Results indicate that the developed HPLC assay can be successfully utilized as a quality control method for simultaneous determination of eight representative substances in Da-Chai-Hu-Tang.

HPLC-Quantification of Diethylamine Salicylate and Methyl Nicotinate in Ointments

Abstract A simple liquid chromatographic method for simultaneous quantification of diethylamine salicylate and methyl nicotinate in the presence of parabens and some drug degradation products is demonstrated. The LC-separation of the two drug substances was undertaken on a column packed with μ-Bondapack C18 using 1% aqueous acetic acid-acetonitrile (85 + 15) at a flow rate of 1.7 ml/min at ambient temperature with detection at 254 nm. Gradient mixing was adopted after 12 min to enhance the rapid elution of the component propyl paraben and other firmly adsorbed ingredients in the ointment base. Good percent assay and mean recoveries with low deviations were obtained for each added drug substance. The stability indicating characteristic of the investigated procedure shows its advantage over another HPLC-method.