Chromatographic Method For Simultaneous Determination Research Articles

Development and Validation of High-Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Curcumin and Artemisinin

Curcumin and Artemisinin are proposed to be formulated as combination therapy to expand the antimalarial effect in resistant and liable isolates. The present research work was to develop and validate a simple, accurate, precise, high-performance thin layer chromatographic method for simultaneous determination of Curcumin and Artemisinin. The chromatographic separation was carried out on a precoated silica gel aluminum plate, 20 × 20 cm, 100μm thickness, with a mixture of Hexane, Toluene and Ethyl acetate (6:1:3) as mobile phase UV detection was performed at 496nm. The Rf were 0.23 and 0.76 for Curcumin and Artemisinin, respectively. Calibration plots were linear over the concentration range 200-1000ng/band for both Curcumin and Artemisinin. The method was validated in accordance with ICH Q2(R1) guidelines for linearity, sensitivity accuracy, precision, and robustness. The percent recoveries in terms of accuracy for the synthetic mixture were found to be 99.8% to 100.0% and 99.94% to 100.4%, and the pooled percent relative standard deviation for repeatability and intermediate precision studies was found to be 0.015-0.042% and 0.008-0.070% for Curcumin and Artemisinin, respectively. A factorial design was used to optimize the chromatographic conditions for the robustness study. In conclusion, a novel, simple, accurate and reproducible HPTLC method was developed and could be applied for quality control testing of Curcumin and Artemisinin in synthetic mixture.

Development of an High Performance Liquid Chromatography Method for Simultaneous Determination of Hesperidin and Sodium Benzoate in Kien Ty Syrup

The Kien ty syrup is formulated from six medicinal materials, including Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, Radix Codonopsis, Rhizoma Atractylodis macrocephalae, Fructus Crataegi, Fructus Hordei germinatus and sodium benzoate as a preservative. Hesperidin, a bioflavonoid found in Pericarpium Citri Reticulatae, Fructus Aurantii immaturus, is used to assess the syrup's quality in terms of assay. In this study, an HPLC method for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup was developed. The method was performed on an Agilent C18 column (250 × 4.6 mm; 5 μm) with the mobile phase being a mixture of 0.05 M phosphate buffer solution pH 5.5: acetonitrile (70:30, v/v), running in a isocratic elution mode. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. The analytes were detected at 225 nm. The method was validated and met the requirements according to ICH, AOAC guidelines. Applying the developed quantification method, the analysis of three sirup lots revealed that the content of hesperidin and sodium benzoate ranged from 0.480-0.504 mg/mL and 2.869-2.306 mg/mL, respectively. The study results indicate that this quantification method can be applied for simultaneous determination of hesperidin and sodium benzoate in Kien ty syrup and similar products.

Development and Validation of High-performance Thin layer Chromatography Method for Simultaneous Determination of Gallic Acid and Quercetin from Annona squamosa L. leaf Extract

The objective of this research was to develop a validated high performance thin layer chromatography (HPTLC) procedure for identification, estimation and standardization of two biomarker compound Quercetin and gallic acid from Annona squamosa L. Leaf hydro alcoholic Extract. The chromatographic quantification were conducted on normal HPTLC (20 cm x 10 cm glass backed silica gel 60 F 254) with Toluene: Ethyl acetate: Formic Acid (5.4:3.8:0.8 v/v/v) used as a mobile phase . The detection and evaluation were performed by densitometric scanning at 254nm. The system was found to give a well resolved, compact and intense peaks for quercetin (Rf 0.45±0.05) and gallic acid ( Rf 0.28±0.05) at 360nm and 280nm respectively from other constituents present in the extract of Annona squamosa L .The proposed analytical method for both the biomarker compound was found to be handy, simple, precise, linear (%RSD =0.673808 and 0.896642 for GA and Quercetin respectively), accurate, reliable and sensitive for analysis of both the biomarkers. The LOD/ LOQ (ng) for Gallic acid and Quercetin were recorded as 15/45 and 12/36, respectively in the linearity range of 200-1200ng per spot. The obtained result showed quantities of Gallic acid and Quercetin in leaf hydroalcoholic extract as 0.023% w/w and 0.048 w/w, respectively. The developed HPTLC was found to be suitable for the routine analysis of both of these biomarkers in the hydroalcoholic extract of Annona squamosa L. and can be further employed in the process quality control of herbal formulations containing the mentioned biomarkers.

A comparative novel green reversed-phase high-performance liquid chromatography method for simultaneous determination of ivabradine and carvedilol in tablets and human plasma using UV and fluorescence detectors

A rapid, green, and sensitive dual detection technique method was applied using both UV and fluorimetric detection to determine ivabradine and carvedilol in their pure form, combined tablet formulation, and in the spiked human plasma. The method was achieved by using HyperClone™ MOS C8 column (150 × 4.6 mm, with particle size 5 μm), and a mixture composed of (70:10:20%v/v) ethanol: methanol: 0.02 M potassium phosphate buffer adjusted to pH = 3 b y using o-phosphoric acid. The dual detection technique was performed at 210 nm in the case of UV detection, and at 337 nm after excitation at 282 nm in the case of fluorimetric detection with a flow rate of 1 mL/min. The retention time in the proposed method was found to be 2.13 and 4.72 min for carvedilol and ivabradine respectively (UV detection) and 2.16 and 4.5 min for carvedilol and ivabradine respectively (fluorescence detection). It was found to be a rectilinear method (r > 0.999), precise (RSD <2 %), and accurate (recoveries 99.92 and 100.52). The method was simple, specific, sensitive, and robust. Calibration graphs were found to be rectilinear from 0.25 to 11.25 μg/mL and 0.50–16 μg/mL for carvedilol and ivabradine for UV detection, while from 30 to 210 ng/mL and 48–400 ng/mL for carvedilol and ivabradine for fluorimetric detection. A correlation coefficient (r) was 0.9999 for the two drugs in UV and fluorimetric detection approaches. Fixed–dose combination tablets containing both medications could be examined routinely by the validated method without interference from excipients. Moreover, the fluorimetric detection HPLC technique can be applied to determine the studied drugs in the human plasma with high accuracy and precision. Greenness of the method was assessed by using Eco-scale and AGREE metrics, and the method was found to be green.

Investigating a Simple and Sensitive High-performance Liquid Chromatography Method for Simultaneous Determination of Apigenin, Catalpol, and Gallic Acid Contents in Plantago Lanceolata L. and Plantago Major L.

Investigating a Simple and Sensitive High-performance Liquid Chromatography Method for Simultaneous Determination of Apigenin, Catalpol, and Gallic Acid Contents in Plantago Lanceolata L. and Plantago Major L.

Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3mL/minute. Separation and analysis were performed on X-bridge C18 (150mm × 4.6mm × 3.5μm) column with diode array detector set at 230nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125μg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62μg/mL for MTR and 0.92 and 2.83μg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time.

Bioanalytical RP-HPLC Method for Simultaneous Quantification of Rivaroxaban and Ticagrelor in Spiked Human Plasma: Validation and Stability

For the effective assessment of preclinical, biopharmaceutical and clinical pharmacological investigations, selective and sensitive analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) are essential. Using Rilpivirine as an internal standard (IS), a simple, fast, sensitive, precise and accurate high performance liquid chromatographic method for simultaneous determination of Rivaroxaban and Ticagrelor in human plasma was developed. The analytes were extracted using a direct protein precipitation method with acetonitrile from 500μL aliquots of human plasma. The content of the drugs was assessed using a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid buffer in a 40:60 v/v ratio and with a flow rate of 1.0ml/min, and an injection volume of 10μL. A Kromasil C18 (250×4.6mm; 5µm) analytical column was used for chromatographic separation and the effluents were monitored at 265nm using a photo diode array (PDA) detector.Rivaroxaban, Ticagrelor and Rilpivirine had retention times of 4.716min, 6.396min and 5.405 min, respectively. Rivaroxaban was shown to be linear at concentrations ranging from 0.018µg/mL - 1.80µg/mL, whereas Ticagrelor was found to be linear between 0.020µg/Ml - 2.00µg/mL. The method was validated according to US-FDA guidelines, and the findings satisfied the acceptance criteria. It was successfully used to determine Rivaroxaban and Ticagrelor in human plasma at the same time and this method is applicable for the pre-clinical and clinical estimation of drugs in biological fluids.

Green liquid chromatographic method for simultaneous determination of metformin, linagliptin and rivaroxaban: Tablet manufacturing and application to in-vitro dissolution studies

Single-pill fixed combination therapy enhances patient adherence and facilitates the effective management of asymptomatic conditions such as cardiovascular disease and diabetes etc. Considering the potential need for a fixed triple-dose combination of metformin HCl, linagliptin and rivaroxaban, here we report, fixed-dose film-coated tablet manufacturing of three co-prescribed drugs (metformin HCl, linagliptin, rivaroxaban) and development of assay method for its analysis followed by in-vitro drug release profile. The analysis was performed under isocratic conditions using phosphate buffer (pH 6.8): ACN (70:30) at 1.00 mL min−1 flow rate. The detector wavelength was adjusted at 248 nm. ICH guidelines were followed for method validation. Linearity was found to be 40–1500, 0.2–7.50 and 0.80–30 µg mL−1 with quantification limits 24.05, 0.264 and 0.565 μg mL−1 for metformin HCl, linagliptin and rivaroxaban respectively. The good reproducibility of the developed method confirmed its suitability for its intended use in routine analyses as per pharmaceutical quality control practices. Greenness evaluation of the proposed method using AES score, AMVI, AGREE, and NEMI tools suggested that the method is environment friendly.

A Novel Validated RP-HPLC Method For the Estimation of Salmeterol By DOE

Background: Salmeterol is a long-acting beta agonist (LABAs) Salmeterol is hypothesized to bind to 2 sites on the beta-2 adrenoceptor. The saligenin moiety binds to the active site of the beta-2 adrenoceptor. The hydrophilic tail of salmeterol binds to leucine residues in the exo-site of the beta-2 adrenoceptor almost irreversibly, allowing salmeterol to persist in the active site, which is responsible for its long duration of action. Method: A new, simple, accurate, rapid, precise, reproducible and cost-effective RP-HPLC method for the quantitative estimation of Salmeterol in bulk and pharmaceutical dosage form. The developed RP-HPLC method for the quantitative estimation of Salmeterol is based on measurement of absorption at maximum wavelength 254 nm using Phosphate buffer: Methanol PH 2.5 (35:65 v/v) as a solvent. The stock solution of Salmeterol was prepared, and subsequent suitable dilution was prepared in mobile phase to obtained standard curve. The standard solution shows absorption maxima at 254 nm. Results: The Salmeterol obeyed Beer Lambert’s law in the concentration range of 20-100μg/ml with regression 0.999 at 254 nm. The overall % recovery was found to be 99.78% for Salmeterol which reflects that the method was free from the interference of the impurities and other excipients, used in the bulk and marketed dosage form. The low value of % RSD was indicative of accuracy and reproducibility of the method. The % RSD for inter-day and intra-day precision was found to be 0.2 for Salmeterol respectively which is &lt;2% hence proved that method is precise. Conclusion: In the present study, novel reverse phase High performance liquid chromatography method for simultaneous determination of Salmeterol in pharmaceutical dosage form was developed. The developed method was validated for various parameters such as accuracy, precision, ruggedness, linearity, robustness, system suitability, specificity as per ICH guidelines.

A Generic High-Performance Liquid Chromatographic Method for Simultaneous Determination of Six Cardiovascular Drugs: Method Optimization and Application to Various Pharmaceutical Formulations

A Generic High-Performance Liquid Chromatographic Method for Simultaneous Determination of Six Cardiovascular Drugs: Method Optimization and Application to Various Pharmaceutical Formulations

Development of the Chromatographic Method for Simultaneous Determination of Azaperone and Azaperol in Animal Kidneys and Livers.

A precise and accurate method for the simultaneous determination of azaperone and azaperol in meat tissues has been developed. This paper describes the first method to be so fast, simple, and useful, especially for many laboratories that do not have sophisticated equipment. This method is based on LC separation and UV-Vis detection. During the sample preparation, the meat tissue was homogenized in acetonitrile at a ratio of 1:4 (tissue weight:acetonitrile volume). The homogenate was centrifuged, the supernatant was evaporated in a lyophilizator, and then the evaporation residue was dissolved in 20 µL of ethanol. For deproteinization, 15 µL of perchloric acid was added, and the sample prepared in this way was injected into a chromatographic column and analyzed using reversed-phased HPLC. The mobile phase consisted of 0.05 mol/L phosphate buffer pH 3.00 (component A) and acetonitrile (component B). UV detection was conducted at 245 nm. The experimentally determined LOQs were 0.25 µg/kg for azaperone and 0.12 µg/kg for azaperol. For both analytes, the calibration curves showed linearity in the tested concentration range from 50 to 300 µg/kg of tissue. The accuracy of the presented method did not exceed 15%, and the recovery was in the range of 85-115%. A validated analytical procedure was implemented for the analysis of various animal tissues for their content of azaperone and azaperol.

Green validated chromatographic methods for simultaneous determination of co-formulated oxytetracycline HCl and flunixin meglumine in the presence of their impurities in different matrices

Green analytical chemistry is an important area of chemical sciences. So, developing competent eco-friendly analytical tools is a big challenge. This study devotes two chromatographic techniques for the simultaneous determination of a binary formulated mixture of oxytetracycline HCl (OXY) and flunixin meglumine (FLU), and their impurities tetracycline (TRC) and 2-chloronicotinic acid (CNA), respectively. Primarily, a TLC densitometric method is proposed and validated using TLC plates sprayed with 5% EDTA of pH 9 as a stationary phase and (acetonitrile/ distilled water/ ethanol 7:2:1, by volume) as a developing system. Well-separated spots are detected at 267 nm where linear relations have been achieved at 0.05–2 μg/band and 0.01–2 μg/band for OXY and FLU, respectively. The second developed and validated method is HPLC which is fulfilled on Hypersil BDS column-C18. A mobile phase of distilled water with 0.1%TFA/ acetonitrile in a ratio of 82: 18, v/v firstly then 70: 30, v/v after 9 min is used in a sequential isocratic elution at 210 nm for FLU and 267 nm for OXY, TRC, and CNA. Sensitive and wide linearity ranges are achieved at 0.05–200 μg/mL for both drugs. The two methods are applied successfully in cattle meat and milk for cited drugs determination. According to ICH guidelines, a validation study has been accomplished for the proposed methods. Statistical comparison has been carried out with official and reported methods. Eventually, the greenness of both procedures is evaluated using Eco-Scale which gives eco-friendly results.Graphical abstract

A reversed-phase-high performance liquid chromatography method for simultaneous determination of paracetamol, caffeine, drotaverine HCl and their related impurities with dissolution profiling of their tablets and greenness profile assessment.

A sensitive, specific and eco-friendly reversed-phase-HPLC method was developed and validated for the determination of paracetamol, caffeine and drotaverine HCl along with their related impurities. The separation was accomplished using an X-bridge C18 column (5 μm; 250 mm × 4.6 mm inner diameter) and a green mobile phase consisting of methanol and 0.02 M phosphate buffer at pH 5.0 in the gradient elution mode. The detector used was a diode array detector. The proposed method was validated in accordance with the International Conference on Harmonisation guidelines. Linear regressions were found in the range of 1-100, 1-100, 2-60, 1-20, 0.50-30 and 1-15 μg/mL for paracetamol, caffeine, drotaverine HCl, p-aminophenol, theophylline and 3,4-dimethoxyphenylacetic acid, respectively. The suggested method was successfully applied for the determination of the studied drugs in their tablet dosage form without interference from any excipients. No discernible difference was found between the obtained results and official or reported methods, statistically, in terms of both accuracy and precision. Dissolution profiling of the studied tablet was also performed using the suggested procedure. Moreover, the greenness profile was assessed using three different tools, namely, the National Environmental Methods Index, the Analytical Eco-Scale and the Analytical GREEnness Metric Approach. The acquired results assert the agreement of the assay with green chemistry principles.

Simple and Rapid High-Performance Liquid Chromatography Method for Simultaneous Determination of Picloram and 2,4-D in Pesticide Formulations

Picloram and 2,4-D are systemic herbicides used to control a wide range of broad-leaved weeds and post-emergence annual and perennial broad-leaved weeds, respectively. The use of plant protection products containing Picloram and 2,4-D necessitates the development of analytical methods, useful for the laboratories focused on control, for monitoring. In this study, we designed and validated an analytical method for the rapid determination of picloram and 2,4-D by HPLC-DAD. The method involves the extraction of the substances by sonication of the sample with methanol, followed by dilution in acetonitrile, and direct injection on a liquid chromatography system, based on the use of a Gemini C18 column. We used an isocratic mobile elution consisting of acetonitrile and water acidified 1% with H3PO4 (50:50, v/v). We validated the proposed method, which demonstrated linearity within the concentration range of 0.01–0.028 mg/mL for picloram and 2,4-D, with a correlation coefficient (R2) of 0.9993 for picloram and 0.9999 for 2,4-D. We considered precision, repeatability and selectivity in the validation. The repeatability of the method expressed as percent of relative standard deviation (%RSD) was lower than 1% for both substances. The proposed method is suitable for the simultaneous determination of picloram and 2,4-D in pesticide formulations.

Validated thin‐layer chromatography method for simultaneous determination of sulfacetamide sodium and hydrocortisone acetate in the ophthalmic formulation

AbstractHigh‐performance thin‐layer chromatography has the utmost separation efficiency. High‐performance thin‐layer chromatography methods can be used to separate, detect, and estimate the different phytochemicals and drug components along with additives. A simple, accurate, precise, and robust high‐performance thin‐layer chromatography method has been developed and validated for the simultaneous estimation of sulfacetamide sodium and hydrocortisone acetate in eye drops. The separation was carried out on pre‐coated silica gel aluminum plate 60 F254 as a stationary phase using ethyl acetate: toluene: methanol: triethylamine (6:2:2:0.1) as a mobile phase. The densitometric measurement of separated components was performed at 254 nm. The method was validated for accuracy, precision, reproducibility, robustness, the limit of detection, and the limit of quantification as per International Council for Harmonization (Q2[R1]) guidelines. The regression coefficients (r2) for sulfacetamide sodium and hydrocortisone acetate were found to be 0.9998 and 0.9994, respectively. The values for percentage recoveries of sulfacetamide sodium and hydrocortisone acetate were found to be 100.2%–100.9% and 101.1%–101.2%, respectively. The developed thin‐layer chromatography method can be used for the analysis of sulfacetamide sodium and hydrocortisone acetate in eye drops.

A pre-column derivatization high-performance liquid chromatography method for simultaneous determination of short-chain and medium-chain fatty acids in a fecal sample.

Short-chain and medium-chain fatty acids have plentiful biological functions, which play a crucial role in the diagnosis and therapy of many diseases. Herein, a new method for simultaneous quantifying 17 short-chain and medium-chain fatty acids with high-performance liquid chromatography coupled with an ultraviolet detector was developed and the pre-column derivatization by indole-3-acetic acid hydrazide was performed to improve the separation and detection. The conditions of the derivatization reaction were systematically investigated. Subsequently, the method was validated and the results showed a satisfactory linearity (linear regression coefficients > 0.9969), the limit of detection (4.0×10-3 -1.9×10-2 μmol/L), precision (0.9%-7.3% for intra-day and 2.0%-9.8% for inter-day), recovery (90.0%-109.1% with relative standard deviation <7.7%) and stability (0.1%-3.3% for standard solution and 0.2%-3.9% for fecal sample). Finally, the established method was successfully applied to quantify short-chain and medium-chain fatty acids in the feces of healthy control and diabetic rats. Eleven kinds of short-chain and medium-chain fatty acids were detected and six of them showed a significant difference between the control group and the model group.

Development and Validation of a High-performance Liquid Chromatography Method for Simultaneous Determination of Five Active Compounds in Kleeb Bua Daeng Formula

Kleeb Bua Daeng formula is a popular traditional remedy sold by the Chao Phya Abhaibhubejhr Hospital, Thailand. This formula contains Piper nigrum L., Nelumbo nucifera Gaertn. and Centella asiatica L. components. A reliable and accurate analytical method for the determination of five major active compounds in this formula was developed and validated. High performance liquid chromatography with a diode-array detector was used to quantify piperine, quercitin, kaempferol, asiaticoside and madecassoside in Kleeb Bua Daeng formula. The separation was carried out using a hypersil C18 column with detection at wavelengths 210, 280 and 370 nm. Gradient chromatographic conditions using a mixture of 0.05% phosphoric acid and acetonitrile allowed for complete resolution of the 5 active compounds. The validation outcomes for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness demonstrated that this HPLC method was accurate and reliable for the simultaneous determination of active compounds in this formula. Hence, this developed and validated HPLC fingerprint method is appropriate for quality control of Kleeb Bua Daeng formula and could be applied to modified Kleeb Bua Daeng formulations.

Green microemulsion electrokinetic chromatographic method for simultaneous determination of azelastine and budesonide

A facile, sensitive, and eco-friendly microemulsion electrokinetic chromatographic method coupled with field amplified sample stacking was developed for the simultaneous determination of azelastine (AZL) and budesonide (BUD) in their combined pharmaceutical preparations. The separation of both drugs was achieved using 13 mM borate buffer (pH 9.0) containing 1.2% sodium dodecyl sulphate (SDS), 2.4% n-butanol, and 1.0% ethyl acetate as the background electrolyte (BGE). A capillary tube (40 cm effective length; 75 μm internal diameter) of uncoated fused silica was used for separation of the studied analytes. A diode array detector was used to monitor the studied compounds at 195 nm. The applied voltage and temperature were adjusted at 28 kV and 25 °C, respectively. The method was linear over the concentration ranges of 0.5–50.0 μg/mL for azelastine and 1.0–20.0 μg/mL for budesonide. The sensitivity was improved by more than 10 folds by using field amplified sample stacking technique. Moreover, the method was applied for separation of budesonide epimers (22R-BUD and 22S-BUD) by increasing the concentration of SDS in the BGE to be 1.8%. Greenness evaluation was performed using analytical eco-scale, Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE) approaches. The excellent green profile of the developed method, confirmed by the three approaches, has supported its applicability in quality control laboratories.

Simultaneous Estimation of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in Combined Dosage Form by HPTLC Method

A simple, step by step, cost-effective, accurate, sensitive, and selective validated High Performance Thin Layer Chromatographic method for simultaneous determination of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in syrup formulation has been developed and validated. HPTLC separation was achieved on Merck aluminum HPTLC plates precoated with silica gel 60 F254. The solvent system comprised of Chloroform: Methanol: Ethyl acetate: Acetic acid: Formic acid (7.0:1.4: 0.8:1.0:0.5 v/v). Densitometric detection wavelength at 200 nm was used in reflectance-absorbance mode. The retention factors were found to be 0.32±0.02, 0.55± 0.02, and 0.72±0.02, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin respectively. Results were found to be linear over a range of 200 - 700ng band-1, 1000 - 7000ng band-1, and 400-1200ng band-1 for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively. The percent assay was found to be 98.47%, 98.47%, and 99.23%, for Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin, respectively in marketed formulation. The developed densitometric method was validated by following the International Council on Harmonisation (ICH) guideline. The developed and validated chromatographic method can be applied for routine quality control of Terbutaline sulphate, Ambroxol hydrochloride, and Guaifenesin in the combined syrup dosage form used in study.

A Simple and Reliable Liquid Chromatographic Method for Simultaneous Determination of Five Benzodiazepine Drugs in Human Plasma

Benzodiazepines (BZDs) are one of the most important drugs that have been used in the treatment of neuropsychological disorders. Indeed, BZDs are abused by drug addicts regardless of their therapeutic uses. Therefore, it was important in forensic and clinical toxicology to reach an easy and reliable method for the screening and quantification of BZDs in the human plasma matrix. In the current work, five BZDs, namely bromazepam, clonazepam, lorazepam, nordiazepam and diazepam were simultaneously separated and detected by a simple and reliable RPLC method in a human plasma matrix. Isocratic mobile elution consisting of 20 mmol L−1 phosphate buffer (pH 7.0) and methanol (50:50, v/v) on a Symmetry C18 column was employed. The flow rate, wavelength and column temperature were fixed at 1.0 mL min−1, 214 nm and 40 °C, respectively. The proposed method was validated, giving a linearity within the concentration ranges 5–500 ng mL−1 for bromazepam and diazepam, 3–500 ng mL−1 for clonazepam and lorazepam and 1–500 ng mL−1 for nordiazepam with a determination coefficient (R2) more than 0.9992. The LOD values for the selected BZDs ranged from 0.54 to 2.32 and from 1.78 to 7.65 ng mL−1 for standard methanolic and plasma matrices, respectively. Precision, accuracy, selectivity, stability, and robustness were some of the terms considered in validating the current RPLC method. Based on these results, a simple and reliable RPLC method was successfully applied to quantify BZDs in human plasma matrix appearing with recoveries ranging from 96.5 to 107.5% and interday RSD less than 4%. The current developed method was useful for rapidly screening the most commonly used BZDs in the market within their therapeutic concentration ranges.