Method For Simultaneous Quantification Research Articles

A rapid and highly sensitive HILIC-HPLC based analytical assay method for quantification of azacitidine and venetoclax in a single run

Venetoclax and Azacitidine combination has recently been approved by the USFDA to treat old people with acute myeloid leukemia. Pharmaceutical formulations of the combination of these two drugs are expected to be launched soon. Till date, an analytical method of any type has not been reported in literature for quantification of Venetoclax and Azacitidine in a single run. The current work set out to establish an HILIC-HPLC based analytical assay method for simultaneous quantification of Azacitidine and Venetoclax from the mixed sample. The method has been developed employing Poroshell HILIC column. Ammonium formate (10 mM, pH 5.0) and acetonitrile were used as a mobile phase in gradient programme. A variety of parameters, including linearity, accuracy, precision, specificity, robustness, and ruggedness were evaluated to validate the method. Azacitidine and Venetoclax were eluted at 8.8 min and 4.0 min, respectively. Method was linear over the range of 1-100 µg/mL. Recovery was found to be 98.29-99.28% and 101.84-101.95% for Azacitidine and Venetoclax, respectively. The method was reproducible with a relative standard deviation of less than two in precision studies. The developed method can suitably be employed for quantifying Venetoclax and Azacitidine in a single run from their mixed samples.

Development and Validation of the HPLC Method for Simultaneous Quantification of Glucose and Xylose in Sugar Cane Bagasse Extract.

A validated rapid high-performance liquid chromatography (HPLC)-refractive index (RI) method was developed for the identification and quantification of glucose and xylose in hydrolyzed bagasse extract. The separation of compounds was achieved on Eurokat® H column (300 × 8mm, 10μm) at 75°C, using 0.01N sulfuric acid solution as mobile phase and 0.6mL/min as flow rate. The method was validated based on accuracy, precision, linearity, robustness, uncertainty, limit of detection (LOD) and limit of quantification (LOQ). Total chromatographic analysis time per sample was ~11min. Calibration plots were linear over the concentration ranges 11-100μg/100μL for glucose and xylose. The LOD was 0.8ppm and LOQ was 2.5ppm. The high recovery and low relative standard deviation confirm the suitability of the method for determination of glucose and xylose in bagasse extract. The proposed HPLC-RI method was accurate, fast and robust and required less run time due to less analytes retention times and allowed optimal energy consumption owing to lower column oven temperature.

Validation of a LC-MS/MS method for simultaneous quantification of 15 free antibiotics in plasma

Validation of a LC-MS/MS method for simultaneous quantification of 15 free antibiotics in plasma

HPLC-Based Metabolomic Analysis and Characterization of Amaranthus cruentus Leaf and Inflorescence Extracts for Their Antidiabetic and Antihypertensive Potential.

The aim of this study was to investigate the potential of Amaranthus cruentus flavonoids (quercetin, kaempferol, catechin, hesperetin, naringenin, hesperidin, and naringin), cinnamic acid derivatives (p-coumaric acid, ferulic acid, and caffeic acid), and benzoic acids (vanillic acid and 4-hydroxybenzoic acid) as antioxidants, antidiabetic, and antihypertensive agents. An analytical method for simultaneous quantification of flavonoids, cinnamic acid derivatives, and benzoic acids for metabolomic analysis of leaves and inflorescences from A. cruentus was developed with HPLC-UV-DAD. Evaluation of linearity, limit of detection, limit of quantitation, precision, and recovery was used to validate the analytical method developed. Maximum total flavonoids contents (5.2 mg/g of lyophilized material) and cinnamic acid derivatives contents (0.6 mg/g of lyophilized material) were found in leaves. Using UV-Vis spectrophotometry, the maximum total betacyanin contents (74.4 mg/g of lyophilized material) and betaxanthin contents (31 mg/g of lyophilized material) were found in inflorescences. The leaf extract showed the highest activity in removing DPPH radicals. In vitro antidiabetic activity of extracts was performed with pancreatic α-glucosidase and intestinal α-amylase, and compared to acarbose. Both extracts exhibited a reduction in enzyme activity from 57 to 74%. Furthermore, the in vivo tests on normoglycemic murine models showed improved glucose homeostasis after sucrose load, which was significantly different from the control. In vitro antihypertensive activity of extracts was performed with angiotensin-converting enzyme and contrasted to captopril; both extracts exhibited a reduction of enzyme activity from 53 to 58%. The leaf extract induced a 45% relaxation in an ex vivo aorta model. In the molecular docking analysis, isoamaranthin and isogomphrenin-I showed predictive binding affinity for α-glucosidases (human maltase-glucoamylase and human sucrase-isomaltase), while catechin displayed binding affinity for human angiotensin-converting enzyme. The data from this study highlights the potential of A. cruentus as a functional food.

Simultaneous Determination of Fipronil, Permethrin and their Key Related Substances in a Topical Drug Product by a Single Stability Indicating High Performance Liquid Chromatography Method.

The topical veterinary drug product containing fipronil and permethrin provides an effective repellent protection and high insecticidal efficacy for dogs. The objective of this study was to develop a stability indicating high performance liquid chromatography (HPLC) method for simultaneous detection and quantification of fipronil, permethrin, their key degradation products and butylated hydroxytoluene (BHT) in a topical drug product. The two active ingredients, their degradation products, and the antioxidant (BHT) were separated by a gradient elution on a Phenomenex Kinetex C18 column (150 × 3 mm, 2.6 µm particle size) maintained at 37 °C with H2O/acetonitrile/isopropyl alcohol/85% H3PO4 (65.5 + 32.5 + 4/0.0053, v/v/v/v) as mobile phase-A and acetonitrile (100%) as mobile phase-B. The flow rate was 0.9 mL/min and analytes were detected and quantified at 235 nm. Specificity of the method was demonstrated by adequate separation of fipronil, permethrin, their degradation products, and BHT in the forced degraded finished product. Linearity of the method was demonstrated in the range of 0.2% to 150% of target analytical concentration of both active ingredients and 50% to 150% for BHT. Excellent recoveries of fipronil, permethrin and BHT in placebo spiked active ingredient solutions in the linearity range showed sufficient accuracy of the method. LOQ and LOD of the method were determined to be 0.2% and 0.07% of the analytical concentration. A robustness study did not identify any critical parameter that adversely effected the separation and quantification. Here, we report the development and validation of a robust, stability indicating HPLC method for identification and assay of fipronil, permethrin, and BHT, including estimation of fipronil's and permethrin's degradation products in a topical drug product for dogs. The new HPLC method permits the acquisition of data for all analytes of interest for a topical finished drug product containing fipronil, permethrin, and BHT.

HPLC Method for Simultaneous Identification and Quantification of Enrofloxacin, Bromhexine*HCl and Their Related Substances/Degradation Products, in Veterinary Pharmaceutical Product, Bromflovet

HPLC Method for Simultaneous Identification and Quantification of Enrofloxacin, Bromhexine*HCl and Their Related Substances/Degradation Products, in Veterinary Pharmaceutical Product, Bromflovet

A validated method for simultaneous quantification of four antiretrovirals in dried blood spot and plasma using LC-MS/MS: Application to efavirenz therapeutic drug monitoring in pregnant patients

IntroductionThe specific physiological background induced by pregnancy leads to significant changes in maternal pharmacokinetics, suggesting potential variability in plasma concentrations of antiretrovirals. Pregnant HIV patients exposed to subtherapeutic doses, particularly in the last trimester of the pregnancy, have higher chances to transmit the infection to their children. Therefore, the therapeutic drug monitoring of antiretrovirals in HIV pregnant patients would be of great value. ObjectivesThis study aimed to develop and validate a sensitive liquid chromatograph tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of efavirenz, raltegravir, atazanavir, and ritonavir in dried blood spots (DBS) and plasma. Design and MethodsThe analytes were extracted from the DBS punch and plasma with a mixture of methanol:zinc sulfate 200 mM (50:50, v/v) and 100 % methanol, respectively. For the chromatographic separation a Shim-pack® C18, 4.6 mm × 150 mm, 5 μm column was used. Detection was performed in a 3200-QTRAP® mass spectrometer, with a run time of 6 min. ResultsThe assay was linear in the range of 15–1,000 ng/mL for raltegravir, 50–10,000 ng/mL for both atazanavir and ritonavir, 50–5,000 ng/mL for efavirenz. Precision and accuracy at these concentrations were less than 15 % for all analytes. Raltegravir, atazanavir, and ritonavir were stable for seven days at 23 °C and 40 °C, whereas efavirenz was stable for twenty-four hours at the same conditions. ConclusionsThe method was successfully applied to quantify efavirenz in DBS samples obtained from HIV-1 infected pregnant volunteers under antiretroviral therapy. The concentrations of efavirenz in DBS and plasma were comparable according to Passing-Bablok regression and Bland-Altman analysis.

Development and validation of a multiclass method for the determination of veterinary drug residues in honey

ABSTRACT This study developed and validated an analytical method for simultaneous identification and quantification of 41 veterinary drugs in honey using liquid liquid extraction (LLE) and liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS). The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at five different concentrations ranging from 0.075 to 50 μg/kg. The mean recoveries ranged from 70 to 105, while repeatability values were all below 17%. The linearity, as correlation coefficients (R2) ranged from 0.994 to 1. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.006–3.92 μg/kg and 0.011–6.54 μg/kg, respectively. The decision limit (CCα) and detection capability (CCβ) ranges were 0.0759–5.33 µg/kg and 0.0804–6.08 µg/kg, respectively. Of the 263 honey samples that were collected from local markets in Egypt, 47.5% had antibiotic residues. The mean concentration (µg/kg) and detection frequency (%) of the five most frequently detected antibiotics in the honey samples were as follows: trimethoprim (143.25 µg/kg and 39.9%), sulfamethoxazole (136.69 µg/kg and 30.7%), sulphadiazine (77.19 µg/kg and 18.6%), tylosin (184.37 µg/kg and 18.2%), and ciprofloxacin (185.33 µg/kg and 7.60%). The applicability of the developed method was proven through successful three proficiency testing (PT). The proposed method was demonstrated to be reliable for the simultaneous analysis of multi-class veterinary drugs in honey.

Robust high-performance thin-layer chromatography (HPTLC) method for stability assessment and simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanoparticles and biological matrices.

Skin cancer poses a significant health risk globally, necessitating effective and safe therapeutic interventions. Epigallocatechin-3-gallate (EGCG) from green tea and rosmarinic acid (RA) from herbs like rosemary offer promising anticancer properties. Combining these compounds may enhance their effectiveness, prompting the need for a reliable analytical method to quantify them. Herein, we present the development and validation of a high-performance thin-layer chromatography (HPTLC) method for concurrent quantification of EGCG and RA in lipid-based nanoparticles and biological samples. The method underwent optimisation through design of experiments (DoE), resulting in the establishment of robust chromatographic conditions. The separation process utilised aluminium HPTLC plates coated with silica gel 60 F254 as the stationary phase, with the mobile phase comprising ethyl acetate, toluene, formic acid, and methanol in a ratio of 4:4:1:1 v/v. The retention factor (Rf) values obtained were 0.38 for EGCG and 0.61 for RA. The method demonstrated linearity over a range of 100-500 ng/band for both compounds with excellent correlation coefficients. Limits of detection and quantification were determined, indicating high sensitivity. Precision evaluations revealed relative standard deviation below 2%, ensuring method reproducibility. Recovery assays in lipid-based nanoparticles, plasma, and urine samples demonstrated excellent recoveries (96.2%-102.1%). Forced degradation studies revealed minimal degradation under various stress conditions, with photolytic degradation showing the least impact. The developed HPTLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents alone and in combination therapies.

A ratiometric electrochemical sensor for simultaneous detection of multiple heavy metal ions in water and herbal medicines based on methylene blue-functionalized metal–organic framework

Heavy metal contamination in water and herbal medicine poses a global threat to human health. There is a pressing need for the development of a simple and rapid method for simultaneous quantification. In this study, a ratiometric electrochemical sensor was first constructed using a hybrid material composed of methylene blue functionalized metal–organic framework (MB-UiO66-NH2) and multi-walled carbon nanotubes (MWCNTs). Then, MB-UiO66-NH2/MWCNTs harnessed the large specific surface area of UiO66-NH2 and good conductivity of MWCNTs, resulting in a sensor with superior analytical performance for Cd2+, Pb2+, and Hg2+ detection. Meanwhile, the ratiometric probe uses MB as an internal reference, with the ratio signal (Imetalion/IMB) serving as output. This ratiometric electrochemical sensor was able to enhance the reproducibility and reliability and eliminate interference, particularly in complex environments. Importantly, based on this ratiometric electrochemical platform, the low detection limit were 11.08 nM for Cd2+, 5.47 nM for Pb2+, and 6.42 nM for Hg2+, which realized the simultaneous, sensitive, and reliable detection. In addition, it has been successfully applied in real water and representative herbal medicine sample analysis. Therefore, this work provides a new approach for the risk assessment of heavy metal accumulation, which is conducive to safeguarding people's lives and health.

Development of a rapid LC-MS/MS method for simultaneous quantification of ten commonly used antibiotic drugs in human serum

The use of TDM in clinical practice to monitor the plasma levels of antibiotics administered to critically ill patients is a well-established approach that allows for optimization of the patient's response to drug therapy, considering the characteristics of the drug, the clinical and physiological status of the patient and any peculiar of the pathogen that caused the clinical picture. In our laboratory, we have developed a single LC-MS/MS analysis for dosing the serum concentration of an antibacterial panel composed of eight antibacterial and two selective inhibitors. The method presented used a certified material furnished by a commercial company and was internally validated using the EMA guidelines. The results have shown high sensitivity, precision, and accuracy, a lower matrix effect combined with simple sample preparation and a time-saving procedure. We have evaluated the recovery rate and matrix effect by testing serum samples without pathological index and serum pools obtained from haemolysed, icteric, or lipemic samples. The assay has shown a recovery range between 94% and 101%.

Identification of Quality Markers in Schisandra chinensis (Turcz.) Baill. Using UPLC-Q-Extractive Orbitrap/MS, Chemometric Analysis, and Network Pharmacology

Chemical composition is a critical factor for determining the efficacy of any traditional Chinese medicine (TCM) and can be used as an indicator of commercial quality. To develop a new strategy for discovering potential quality markers (Q-markers) of TCM by integrating ultra-performance liquid chromatography-Q-extractive orbitrap/mass spectrometry (UPLC-Q-Extractive Orbitrap/MS), chemometric analysis, and network pharmacology, using Schisandra chinensis (Turcz.) Baill. (S. chinensis) as an example. The chemical profiling of S. chinensis was performed using UPLC-Q-Extractive Orbitrap/MS, followed by identification of hepatoprotective Q-markers through a comprehensive understanding of chemometric analysis and virtual target prediction of network pharmacology. Six compounds were considered potent candidates for Q-markers, which were identified as schisandrol A (6), angeloylgomisin H (10), schisantherin A (17), schisantherin B (18), schisandrin A (23), and schisandrin C (26). All Q-markers exhibited significant hepatoprotective activity, as evidenced by in vitro experiments. Subsequently, a method for simultaneous quantification was established and employed to analyse seven batches of S. chinensis. Therefore, the integrated approach of UPLC-Q-Extractive Orbitrap/MS, chemometrics, and network pharmacology proved to be an effective strategy for the discovery of Q-markers that can assist in assessing the overall chemical consistency of samples and provide a basis for quality evaluation of the material basis of S. chinensis.

“Plant Golden” C. sativus: Qualitative and quantitative analysis of major components in stigmas and petals and their biological activity in vitro

Crocus sativus L. (C. sativus) has its stigma as the main valuable part used. With extremely low production and high prices, stigma is considered a scarce resource. As a result, its petals, considered as by-products, are often discarded, leading to significant waste. We developed a UPLC-Q-Orbitrap HRMS method for qualitative analysis of stigmas and petals and a UHPLC-QQQ-MS/MS method for simultaneous quantification of 9 characteristic active compounds for the first time, and compared their biological activity in vitro. The results indicated that a total of 63 compounds were identified in the petals and stigmas. The content of flavonoids in the petals was significantly superior to that in the stigma, and the content of quercetin in the petals was 50 times higher than that in the stigma. The results of the in vitro evaluation of biological activity indicated that both the petals （•OH: IC50=39.70 mg/mL; DPPH: IC50=28.37 mg/mL; ABTS: IC50=0.9868 mg/mL）and stigma （•OH: IC50=34.41 mg/mL; DPPH: IC50=38.99 mg/mL; ABTS: IC50=3.194 mg/mL）demonstrated comparable antioxidant activities. However, the tyrosinase inhibitory activity in petals （IC50=21.17 mg/mL） was weaker than that in stigma（IC50=1.488 mg/mL）. This study provides a fast, reliable, and efficient analytical method that can be used for the quality assessment of petals as a natural resource and its related products in the food and pharmaceutical industries.

Development and Validation of a RP-HPLC Method for Simultaneous Quantification of Paracetamol and Phenylephrine Hydrochloride

Development and Validation of a RP-HPLC Method for Simultaneous Quantification of Paracetamol and Phenylephrine Hydrochloride

A validated, precise TLC-densitometry method for simultaneous quantification of mahanimbine and koenimbine in marketed herbal formulations

BackgroundMurraya koenigii (L.) Spreng. (Rutaceae) is an important medicinal plant in natural products research for its diverse pharmacological activities. Carbazole alkaloids were the major classes of phytoconstituents obtained from different parts of this plant, such as leaves, stems, and roots. Mahanimbine and koenimbine are two important carbazole alkaloids obtained from the M. koenigii plant and are known for their anti-cancer, anti-oxidant, anti-diarrhoeal agents, etc. Standardization plays a vital role in the herbal drug industry for maintaining the quality, purity, safety, and efficacy of herbal formulations, and hyphenated analytical techniques like HPTLC, HPLC, GC–MS, and LC–MS were utilized for this purpose. In the present study, a specific, simple, and rapid semi-automated TLC method was developed to quantify mahanimbine and koenimbine in some marketed herbal formulations, and the same was validated based on (ICH)-Q2-(R1) guidelines.ResultsThis study revealed that the powder formulation (F3) contains the highest amount of mahanimbine (62.32 µg), but the tablet formulation (F1) contains both mahanimbine (41.19 µg) and koenimbine (143.6 µg).ConclusionA simple, specific, and reproducible semi-automated TLC method was developed and validated successfully as per (ICH)-Q2-(R1) guidelines and can be utilized for analysing marketed herbal formulations containing M. koenigii powder/extracts.

Development of simultaneous quantitation method for 20 free advanced glycation end products using UPLC–MS/MS and clinical application in kidney injury

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93–5.94 % and 2.30–8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1–3.13 ng/mL and 0.5–6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R2 >0.9974) across a wide linear range (1.56–400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N6-carboxyethyl-L-arginine (CEA), N6-carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N6-formyl-lysine, N6-carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease.

Development of a Simultaneous Quantification Method for Multiple Modes of Nitrogen in Leaf Models Using Near-Infrared Spectroscopic Measurement.

By focusing our attention on nitrogen components in plants, which are important for cultivation management in data-driven agriculture, we developed a simple, rapid, non-chemical and simultaneous quantification method for proteinic and nitrate nitrogen in a leaf model based on near-infrared (NIR) spectroscopic information obtained using a compact Fourier Transform NIR (FT-NIR) spectrometer. The NIR spectra of wet leaf models impregnated with a protein-nitric acid mixed solution and a dry leaf model obtained by drying filter paper were acquired. For spectral acquisition, a compact MEMS (Micro Electro Mechanical Systems) FT-NIR spectrometer equipped with a diffuse reflectance probe accessory was used. Partial least square regression analysis was performed using the spectral information of the extracted absorption bands based on the determination coefficients between the spectral absorption intensities and the contents of the two-dimensional spectral analysis between NIR and mid-infrared spectral information. Proteinic nitrogen content in the dry leaf model was well predicted using the MEMS FT-NIR spectroscopic method. Additionally, nitrate nitrogen in the dry leaf model was also determined by the provided method, but the necessity of adding the data for a wider range of nitric acid concentrations was experimentally indicated for the prediction of nitrate nitrogen content in the wet leaf model. Consequently, these results experimentally suggest the possibility of the application of the compact MEMS FT-NIR for obtaining the bioinformation of crops at agricultural on-sites.

Development of the RP-HPLC Method for Simultaneous Determination and Quantification of Artemether and Lumefantrine in Fixed-Dose Combination Pharmaceutical Dosage Forms.

Developing countries face enormous challenges with substandard and falsified antimalarial drugs. One specific issue is the lack of a simple, cost-effective, and robust HPLC method to simultaneously determine and quantify the active pharmaceutical ingredients (APIs) in fixed-dose artemether-lumefantrine pharmaceutical dosage forms. The current study developed a novel, simple, sensitive, precise, accurate, and cost-effective RP-HPLC method for the simultaneous determination and quantification of artemether and lumefantrine in pharmaceutical dosage forms. The HPLC analysis was carried out on an Agilent 1260 Infinity Series HPLC system equipped with an ODS Intersil-C8 (150 × 4.6 mm) 5.0 µm column, by isocratic elution. The mobile phase composition consisted of acetonitrile and 0.05% orthophosphoric acid buffer of pH 3.5 in the ratio of 70 : 30 v/v. The analysis was performed at a 1 mL/min flow rate and a column temperature of 25°C. The total run time was 6 minutes. The detection was done with a variable wavelength detector (VWD) at an isosbestic point wavelength (λ) of 210 nm. The developed method was validated according to the ICH guidelines concerning system suitability, specificity, linearity, accuracy, precision, and robustness. The system suitability of the developed method revealed satisfactory theoretical plates and symmetry factors. The method proved to be specific, with no interference of mobile phase or excipients. The calibration plot exhibited linearity over the concentration range of 275-1925 μg/mL with R2 = 0.9992 for artemether and a range of 150-1050 μg/mL with R2 = 0.9985 for lumefantrine. The accuracy of the method, determined by the recovery study, was 99.79-100.16% for artemether and 99.04-99.50% for lumefantrine. The % RSD values for intraday precision were 0.175 and 0.203, while interday precision values were 0.340 and 0.554 for artemether and lumefantrine, respectively. The method demonstrated robustness when subjected to slight modifications in the flow rate, column temperature, and mobile phase composition. The developed analytical method proved satisfactory as per ICH guidelines and hence can be used for the determination and quantification of artemether and lumefantrine in bulk drug and pharmaceutical dosage forms.

A Validated RP-HPLC Method for Simultaneous Quantification of Abacavir and Zidovudine and their Impurities in Combined Dosage Forms

Develop a simple, accurate, and precise method for simultaneous analysis of Abacavir and Zidovudine, along with their related impurities, in combined pharmaceutical dosage forms. A reverse- phase high-performance liquid chromatography (RP-HPLC) method was developed using an LC-20 AT C18 column and a Water: Methanol (80:20) mobile phase. Detection was carried out at 240 nm. The method successfully separated Abacavir, Zidovudine, and their respective impurities with distinct retention times. Validation demonstrated good linearity, accuracy, and precision for both drugs and their impurities. Limits of detection (LOD) and quantification (LOQ) were determined and found to be suitable for analyzing impurities in commercial dosage forms. The developed RP-HPLC method offers a simple, accurate, and reliable tool for simultaneous quantification of Abacavir, Zidovudine, and their impurities in combined dosage forms, thereby contributing to quality control and ensuring drug safety. Keywords Abacavir, Zidovudine, Related Substance RP-HPLC Method, ICH Q2 (R1) guidelines

An isocratic RP-HPLC-UV method for simultaneous quantification of tizanidine and lidocaine: application to in vitro release studies of a subcutaneous implant.

Implantable devices have been widely investigated to improve the treatment of multiple diseases. Even with low drug loadings, these devices can achieve effective delivery and increase patient compliance by minimizing potential side effects, consequently enhancing the quality of life of the patients. Moreover, multi-drug products are emerging in the pharmaceutical field, capable of treating more than one ailment concurrently. Therefore, a simple analytical method is essential for detecting and quantifying different analytes used in formulation development and evaluation. Here, we present, for the first time, an isocratic method for tizanidine hydrochloride (TZ) and lidocaine (LD) loaded into a subcutaneous implant, utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a UV detector. These implants have the potential to treat muscular spasticity while providing pain relief for several days after implantation. Chromatographic separation of the two drugs was accomplished using a C18 column, with a mobile phase consisting of 0.1% TFA in water and MeOH in a 58 : 42 ratio, flowing at 0.7 ml min-1. The method exhibited specificity and robustness, providing accurate and precise results. It displayed linearity within the range of 0.79 to 100 μg ml-1, with an R2 value of 1 for the simultaneous analysis of TZ and LD. The developed method demonstrated selectivity, offering limits of detection and quantification of 0.16 and 0.49 μg ml-1 for TZ, and 0.30 and 0.93 μg ml-1 for LD, respectively. Furthermore, the solution containing both TZ and LD proved stable under various storage conditions. While this study applied the method to assess an implant device, it has broader applicability for analysing and quantifying the in vitro drug release of TZ and LD from diverse dosage forms in preclinical settings.