Compounds In Pharmaceutical Formulations Research Articles

Quality by Design Approach Driven Development and Validation of Reverse Phase‐High‐Performance Liquid Chromatography Methodology for Dual Quantification of Aspirin and Rivaroxaban

ABSTRACTClinical trials have shown that combining a modest dose of Rivaroxaban (RIV) with Aspirin (ASP) is more effective than ASP alone in reducing cardiovascular mortality, myocardial infarction, and stroke. A high‐performance liquid chromatography method developed using Quality by Design (QbD) effectively estimates ASP and RIV for routine pharmaceutical analysis. The Plackett‐Burman design assessed key factors including organic phase volume, mobile phase pH, flow rate, temperature, and injection volume using an Ishikawa fish‐bone diagram for risk assessment. These parameters were then optimized via Box‐Behnken design in 15 trials. The method meets the International Council For Harmonization standards with a mobile phase ratio of 45:55 (v/v) acetonitrile: buffer (pH 3, adjusted with orthophosphoric acid), a flow rate of 1 mL/min, a column temperature of 30°C, and an injection volume of 20 µL. A Phenomenex Luna C18 column (250 × 4.6 mm, 5 µm particle size) with detection at 251 nm ultra‐violet wavelength. The retention times of 4.54 min for ASP and 5.81 min for RIV were obtained. Validation confirmed linear ranges: 40–200 µg/mL for ASP and 2–10 µg/mL for RIV, with excellent recovery rates (ASP: 99.30%–101.9%; RIV: 98.52%–101.84%). Developed using QbD principles, the method proved specific, accurate, precise, sensitive, and robust for routine quality control of both compounds in pharmaceutical formulations.

Quantification of oxaliplatin- and ioversol-related compounds in pharmaceutical formulations using novel HPLC-ICP-MS methods

PurposeAccurate quantifying of drug-related compounds in medicines is vital for safety. Commonly used structure-dependent methods rely on analytical standards. High-performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) offers a promising solution, being structure-independent and not requiring standards. In this study, we aim to develop HPLC-ICP-MS methods for the determination of related compounds in oxaliplatin and ioversol injections. ResultsThe target analytes were eluted on an XSelect HSS T3 column (2.1 ×50 mm, 5 µm). Specifically, oxaliplatin injection was eluted isocracially for 3.5 min, and ioversol injection was eluted gradient with a total chromatographic run time of 12 min. The measurements to determine dihydroxy oxaliplatin-Pt(IV) and two related compounds of ioversol were performed by monitoring at m/z for 195Pt and 127I, respectively. The calibration curves were established over the range of 0.05–1 μM for Pt and 0.3–15 μM for I with the correlation coefficients greater than 0.999. The limits of quantification were 0.004 μM for dihydroxy oxaliplatin-Pt(IV), 0.022 μM for ioversol related compound A and 0.026 μM for ioversol related compound B. The accuracy (recovery between 93–105%) and precision (repeatability ≤ 6.1% RSD) were fit-for-purpose for dihydroxy oxaliplatin-Pt(IV), and the accuracy (recovery between 95–107%) and precision (repeatability ≤ 3.9% RSD) were also fit-for-purpose for both ioversol related compound A and ioversol related compound B. ConclusionThe quantitation accuracy of HPLC-ICP-MS closely matched that of the standard HPLC-UV approach. HPLC-ICP-MS can be used as a complementary analytical technique for quantitative determination of drug-related compounds.

Assessing Medicinal Plant Extracts in Strengthening Anti-Dandruff Shampoo: An Analysis Using Multi-Criteria Decision-Making Techniques

Currently, more than half of the global population grapples with dandruff. The presence of the Malassezia fungus significantly contributes to this condition by potentially triggering the production of cytokines in keratinocytes, the cells responsible for synthesizing keratin and activating inflammatory pathways. Given the distress caused by dandruff, considerable efforts are underway to find effective treatments. Numerous studies centered on medicinal plants suggest promising efficacy in addressing this issue. This particular research focuses on conducting a clinical trial to explore the effectiveness of zinc L-pyrrolidone carboxylic acid and pirocton olamine, combined with extracts from six medicinal plants, for dandruff therapy. In this study, a combination of methanolic extracts from alternatives, along with pirocton olamine and zinc-PCA in the form of shampoo, was applied to a group of 30 individuals experiencing dandruff. This treatment regimen was carried out over a span of two months to assess its effectiveness in managing dandruff. Notably, 15 patients with persistent dandruff experienced significant relief within the initial two weeks. Another 12 patients noticed dandruff reduction after 28 days, the majority experienced relief within the first few weeks, and by the end of the fifth week, those remaining also expressed satisfaction with the treatment. These results strongly highlight the impressive effectiveness of medicinal plant extracts in alleviating dandruff, accompanied by minimal side effects, albeit requiring extended treatment duration. The research indicates that blending medicinal plant extracts with both natural and chemical compounds in pharmaceutical formulations shows potential for enhancing the effectiveness of dandruff treatments.

Development of a robust and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method using analytical quality by design principles for the accurate determination of esculin in its bulk form

BackgroundAnalytical quality by design is a proactive, holistic, and data-driven approach to quality that emphasizes risk assessment and management. This can lead to more robust and reliable methods than traditional approaches. Using principles of analytical quality by design for method development can help to assure the quality and consistency of analytical methods. This is important for the pharmaceutical industry, where accurate and reproducible analytical methods are essential for ensuring drug safety, shelf life, and efficacy. Esculin is a naturally occurring derivative of coumarin that is found in the stems of the plant Aesculus indica. The present study describes the use of an analytical quality by design approach to develop and validate a reliable RP-HPLC method for the analysis of esculin bulk form.ResultA central composite design was employed to optimize the percent of methanol in the mobile phase and flow rate for the analysis of a compound esculin using the RP-HPLC method. The optimized conditions were 43% methanol and 0.9 ml/min flow rate, with a retention time of 3.78 min, and Phenomenex Luna (5 µm × 250 mm, 4.6 mm) column was used. The method was found to be linear with a correlation coefficient of 0.9998 for a concentration range of 4–20 μg/ml. The parameters of the system suitability test were within the acceptable range (0.0612–0.1398%), and the precision for both intra-day and inter-day measurements was below 2%. The robustness and ruggedness of the method were also good, with changes in the flow rate and mobile phase composition having a minimal impact on the method's performance. The limit of detection (LOD) and limit of quantification (LOQ) values were reported to be 0.82891 μg/ml and 2.511 μg/ml, respectively. The validation parameters of the method adhered to the specified limit following the ICH guidelines.ConclusionIn summary, an AQbD-based efficient and robust RP-HPLC chromatographic method has been developed for the quantification of the esculin compound. The method is linear, precise, and reproducible, and it has good LOD and LOQ values. The method could be used for repetitive analysis of the compound in pharmaceutical formulations.

Zein nanoparticles stabilized by hydrophilic small molecule stabilizer matrine deliver curcumin effectively

Matrine (MAR), a quinolone alkaloid, was employed to augment the stability of zein nanoparticles. The incorporation of MAR into the hydrophobic shell of zein nanoparticles was primarily achieved through hydrogen bonding. Curcumin (CUR), a hydrophobic active substance, was encapsulated in the hydrophobic core of zein/matrine nanoparticles (ZMNPs). The preparation of ZMNPs and curcumin-loaded zein/matrine nanoparticles (CZMNPs) was accomplished using an antisolvent precipitation method. The encapsulation efficiency of curcumin in ZMNPs (zein/MAR = 8:1, 20 mg zein and 2.5 mg matrine) was significantly greater (52.64%) than that of nanoparticles produced from a single zein (2.50%). CZMNPs demonstrated a notable encapsulation efficiency and loading capacity (88.30% and 7.84%, respectively) upon the addition of 2 mg of curcumin, and were capable of sustained and gradual release of curcumin in simulated intestinal fluid. Furthermore, the stability of ZMNPs was observed to be favorable across a range of environmental conditions, including pH levels of 2–4 and 6–9, salt concentrations of ≤150 mM, temperatures of ≤90 °C, and storage at room temperature for a duration of 30 days. Additionally, the inherent anti-cancer properties of MAR make CZMNPs a more efficacious inhibitor of tumor cell proliferation in vitro. Moreover, the uptake of CZMNPs by A549 cells was significantly enhanced, potentially through the process of endocytosis. Therefore, the incorporation of matrine in zein-based nanoparticles confers anticancer properties to the resulting ZMNPs. These nanoparticles can serve as encapsulating agents for bioactive compounds in pharmaceutical formulations and as a novel delivery strategy for long-term cancer care. Specifically, matrine is anticipated to function as a potential stabilizer for other nanosystems.

Chemometric Tools in the Analysis of Pharmaceutics Samples: a Comparison Among Several Multivariate Calibration Methods

Bivariate calibration algorithm is compared with the results obtained by the usage of high-dimensional calibration methods such as partial least squares (PLS) and multi-way partial least-squares (N-PLS) by using UV-Vis spectrophotometric data of first and second-order. The algorithms were applied to the determination of a mixture of an analgesic and a stimulant compound and their actual concentrations of them were calculated by using spectroscopic data. The direct reading of absorbance values at 227 nm and 271 nm were employed for quantification of the compounds in the case of the bivariate method. The approaches of first-order and multi-way methods were applied with a previous optimization of the calibration matrix by constructing sets of calibration and validation with 20 and 10 samples (mixtures) respectively according to a central composite design and their UV absorption spectra were recorded at 200-350 nm. All algorithms were satisfactorily applied to the simultaneous determination of these compounds in pharmaceutical formulations with mean percentage recovery of 100.5 ± 3.67, 98.7 ± 3.42, and 100.5 ± 3.74 for bivariate, PLS-1, and N-PLS, respectively. The statistical evaluation of the bivariate method showed that this procedure is comparable with those algorithms that employ high-dimensional structured information. The aim of the work is to compare the methods under study and it can be seen that there are no significant differences, so a simple spectrophotometer can be used up to a very specialized one. However, the advantage of bivariate calibration is its simplicity, due to the minimal experimental manipulation.

Selection of essential spectra to improve the multivariate curve resolution of minor compounds in complex pharmaceutical formulations

Multivariate curve resolution unmixing of hyperspectral imaging data can be challenging when low sources of variance are present in complex samples, as for minor (low-concentrated) chemical compounds in pharmaceutical formulations. In this work, it was shown how the reduction of hyperspectral imaging data matrices through the selection of essential spectra can be crucial for the analysis of complex unknown pharmaceutical formulation applying Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS). Results were obtained on simulated datasets and on real FT-IR and Raman hyperspectral images of both genuine and falsified tablets. When simulating the presence of minor compounds, different situations were investigated considering the presence of single pixels of pure composition as well as binary and ternary mixtures. The comparison of the results obtained applying MCR-ALS on the reduced data matrices with those obtained on the full matrices revealed unequivocal: more accurate decomposition could be achieved when only essential spectra were analyzed. Indeed, when analyzing the full dataset, MCR-ALS failed resolving minor compounds even though pure spectra were provided as initial estimation, as shown for Raman hyperspectral imaging data obtained on a medicine sample containing 7 chemical compounds. In contrast, when considering the reduced dataset, all minor contributions (down to 1 pixel over 17,956) were successfully unmixed. The same conclusion could be drawn from the results obtained analysing FT-IR hyperspectral imaging data of a falsified medicine.

Analytical capabilities of micellar liquid chromatography and application to residue and contaminant analysis: A review.

Green chemistry is the use of chemistry to reduce or eliminate the use of generation of feedstocks, products, by-products, solvents, reagents, etc. that are hazardous to human health or the environment. One of the branches of green chemistry is micellar liquid chromatography. Micellar liquid chromatography is a reversed-phase liquid chromatographic mode with mobile phases containing a surfactant above its critical micellar concentration. The applications of micellar liquid chromatography for the determination of numerous compounds in pharmaceutical formulation, biological samples, food, environmental samples, and feeds have been growing rapidly. Micellar liquid chromatography technique has several advantages over other chromatographic techniques. Its main advantage is the small amount of organic modifiers used such as acetonitrile and methanol and the safety and recyclability of the mobile phase. In our work, we discuss the development of "green chemistry" and present what micellar liquid chromatography is. This article presents application methods with the use of micellar liquid chromatography for analysis on antibacterial substances, melamine, biogenic amines, plant protection products, flavonoids, as well as peptides in biological matrices such as milk, eggs, tissues, honey, and feed.

Anti-Helicobacter pylori Compounds from Oliveria decumbens Vent. through Urease Inhibitory In-vitro and In-silico Studies.

Oliveria decumbens Vent. has been used by indigenous people of southwest Iran for treating peptic ulcers and gastrointestinal infections. This study aimed to investigate the antibacterial activity of Oliveria decumbens extract and fractions and to analyze the bioactive components of the fractions. Total plant extract and different fractions of Oliveria decumbens Vent. were prepared. Antibacterial activities were evaluated against the clinical strain of Helicobacter pylori and standard strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa using agar dilution and disc diffusion methods. Phytochemical analysis of the fractions was performed using silica gel chromatography and 1D and 2D NMR spectroscopy. Moreover, the urease inhibitory effects of the isolated compounds were assessed in-vitro and in-silico. Three novel kaempferol derivatives and two thymol derivatives were isolated from Oliveria decumbens aerial parts, and the structures were determined by comparison with published data. The n-hexane fraction was found to exert the most significant anti-H. pylori activity with the minimum inhibitory concentration of 50 µg/mL. All fractions demonstrated antibacterial activity toward S. aureus. In-vitro urease inhibition assay showed that stigmasterol, tiliroside, and carvacrol were found to be the most potent enzyme inhibitors in the isolated compounds. Molecular interactions of the compounds with the active site of urease were supported by the molecular docking analysis. Novel bioactive compounds in Oliveria decumbens were described in this study. The antibacterial effects suggested the potential use of the compounds in pharmaceutical formulations inconsistent with the traditional use of the plant in the treatment of gastrointestinal infections.

Rapid Raman Spectroscopic Analysis of Stress Induced Degradation of the Pharmaceutical Drug Tetracycline.

Stress factors caused by inadequate storage can induce the unwanted degradation of active compounds in pharmaceutical formulations. Resonance Raman spectroscopy is presented as an analytical tool for rapid monitoring of small concentration changes of tetracycline and the metabolite 4˗epianhydrotetracycline. These degradation processes were experimentally induced by changes in temperature, humidity, and irradiation with visible light over a time period of up to 23 days. The excitation wavelength λexc = 413 nm was proven to provide short acquisition times for the simultaneous Raman spectroscopic detection of the degradation of tetracycline and production of its impurity in small sample volumes. Small concentration changes could be detected (down to 1.4% for tetracycline and 0.3% for 4-epianhydrotetracycline), which shows the potential of resonance Raman spectroscopy for analyzing the decomposition of pharmaceutical products.

Spectrophotometric Determination of Amoxicillin, Ampicillin, Cefalexin and Cefadroxil in Pharmaceutical Formulations, Biological Fluids and Spiked Water Samples

Amoxicillin, ampicillin, cefalexin and cefadroxil are the most extensively used beta-lactam antibiotics due to their broad spectrum of action and low cost. The quality of the pharmaceutical formulations of these drugs being marketed needs to be ensured to achieve a better remedial effect and a lower toxicity. The widespread exploitation of antibiotics sometimes leads to their presence in residues in body fluids and water bodies, which is potentially toxic and dangerous for human health. Thus, the determination of these drug compounds in pharmaceutical formulations, biological fluids and in water samples is of great importance and scope. Owing to the inherent ease and simplicity and sensitivity of spectrophotometric methods, the reactions of above drug compounds with carbon disulphide and copper(I) perchlorate in the presence of pyridine to form acrylonitrile extractable yellow coloured copper(I) drug dithiocarbamate complexes has been investigated for the spectrophotometric determination of these drugs. The analysis is accomplished by measuring the intensity of yellow extract at 450, 378, 380 and 430 nm for amoxicillin, ampicillin, cefalexin and cefadroxil respectively against a reagent blank. Under the optimized condition calibration graphs were linear in the ranges of 1.68-167.78, 1.61-161.38, 1.39-138.96 and 1.45-145.36 μg/mL with the detection limit (3.3 σ / s) of 1.24, 1.19, 1.07 and 1.13 μg/mL respectively. The high recoveries of the listed drugs by the proposed method from respective pharmaceutical formulations (98.80-100.30 %), simulated biological fluids (98.46-100.21 %) and spiked water samples (98.32-99.66 %) with RSDs in the ranges 0.15-1.11 % indicate good accuracy and precision of the method.

Bi2O3/1-butyl-1-methylpyrrolidinium bis (trifluoromethane sulfonyl) imide glassy carbon platform: strategy for the electrocatalytic ultrasensitive quantification of multianalytes

In the present communication studies on glassy carbon electrode (GCE), modified with Bi2O3 metal oxide and 1-butyl-1-methylpyrrolidinium bis (trifluoromethane sulfonyl) imide ionic liquid (IL) (Bi2O3/IL/GC), sensor for the voltammetric investigation of ethamsylate (ETA) and mefenamic acid (MEF) simultaneously has been incorporated. The developed sensor was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV). All the analytical parameters were also examined during the electrochemical measurements of these compounds and experimental conditions were optimized. The chemically modified sensor exhibited two well-resolved oxidation peaks at 0.54 and 0.79 V for ETA and MEF, respectively, in phosphate buffer pH 2.0. A good linear response was achieved within the concentration range from 15 to 105 nM for ETA and 1 to 7 nM for MEF. The limit of detection (LOD) for ETA was 6.3 nM and for MEF was 0.4 nM, and limit of quantification (LOQ) for ETA and MEF was 19 nM and 1.2 nM respectively. The developed sensor could be applied to quantify the concentrations of both the compounds in pharmaceutical formulation and human serum sample with satisfactory results.

Micellar Liquid Chromatography from Green Analysis Perspective

Abstract Micellar liquid chromatography (MLC) is a simple well-established branch of high-performance liquid chromatography. The applications of MLC for the determination of numerous compounds in pharmaceutical formulations, biological samples, food, and environmental samples have been growing very rapidly. MLC technique has several advantages over other techniques, such as simultaneous separation of charged and uncharged solutes, rapid gradient capability, direct on-column injection of physiological fluids, unique separation selectivity, high reproducibility, robustness, enhanced luminescence detection, low cost, and safety. This review is devoted to the evaluation of the agreement of MLC with the principles of green chemistry which recently represents a universal trend. Also, it provides an overview on the basics of MLC, in addition to a survey of MLC methods published in the past five years for the assay of various compounds in different matrices.

Chemometrics-Assisted UV Spectrophotometric Method for Determination of Metformin Hydrochloride and Glyburide in Pharmaceutical Tablets

This study aims to estimate simultaneously metformin hydrochloride (MET) and glyburide (GLY) in a multicomponent tablets dosage form by spectrophotometric method using chemometric approaches such as principal component regression (PCR) and partial least-squares regression (PLS). Because of highly overlapped in UV spectra and difference proportions of two active ingredients, the conventional univariate calibration methods was not allowed without previous separation. The linearity ranges used to construct the calibration matrix were selected in the ranges from 40.00 to 200.00 mg L-1for MET and from 1.00 to 10.00 mg L-1for GLY. The absorbances were measured in the wavelength range of 200-400 nm, using ethanol as solvent. The resulting UV spectra were subjected to PCR and PLS algorithms and the optimum numbers of principal components (PCs) were selected according to prediction residual error sum of squares (PRESS) values of leave-one out cross-validation. The number of PCs for MET and GLY were found to be 5, 3 by PCR and 5, 3 by PLS, respectively. A set of synthetic mixtures was employed to verify the models and the performance of the models were shown in the values of the root mean square error in prediction (RMSEP). RMSEP values of MET and GLY were 1.806, 0.256 for PCR and 1.802, 0.185 for PLS, respectively. The suitable calibration models were applied to the analysis of these compounds in pharmaceutical formulation.

Ultra-fast determination of caffeine, dipyrone, and acetylsalicylic acid by capillary electrophoresis with capacitively coupled contactless conductivity detection and identification of degradation products

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) was used for fast, simultaneous determination of dipyrone (DIP), caffeine (CAF), and acetylsalicylic acid (ASA). In the same run and in less than 1min, the degradation products from DIP and ASA were also detected. In addition, the usage of the CE-C4D system allowed, for the first time, the detection of methylamine as a degradation product of DIP. Capillary electrophoresis with electrospray mass spectrometry experiments were carried out in order to confirm the formation of methylamine. The limits of detection by CE-C4D were 5, 5, and 6μmolL−1 for CAF, DIP, and ASA, respectively. The proposed method was applied to the analysis of these compounds in pharmaceutical formulations with similar results to those achieved by HPLC (p<0.05).

Recent Advances in the Use of Cyclodextrins in Antifungal Formulations

Cyclodextrins are usually used in antifungal formulations as auxiliary substances to improve solubility, stability, or other physicochemical properties of the active compound. Nevertheless, more and more research and practical use results indicate that cyclodextrins might also act as active compounds in pharmaceutical formulations. The biological effects of cyclodextrins, important for their use within antimycotic formulations, can be divided into: effects based on the ability of cyclodextrins to form inclusion complexes with endogenous substances (membrane lipids, cellular cholesterol), effects based on formation of inclusion complexes with component parts of fungi cells, and effects based on the chemical nature of cyclodextrins and their derivatives. This review will cover the advances in research of biological activity of cyclodextrins with focus on their properties responsible for their synergistic effect with antimycotic compounds.

Electrochemistry of olanzapine and risperidone at carbon nanotubes modified gold electrode through classical and DFT approaches

Among the neuroleptics, atypical antipsychotics have received great attention in recent years, because of their peculiar pharmacological profile. Due to their widespread use, rapid, efficient and economical methods of analysis of these compounds in pharmaceutical formulations and biological fluids are required, and in this sense electrochemical techniques appear particularly attractive. Most of these compounds are reported to be electroactive but quantification at classic electrodes is often unsuitable due to electrode fouling and absence or reduced linearity between concentration and current intensity. A gold electrode modified with oxidized single walled carbon nanotubes has proved useful to overcome these problems. A deep insight into the electrochemistry of these compounds is reported, with the aid of Mass Spectrometry and DFT (Density Functional Theory) calculation; in particular, a computational approach to the electrochemistry of these substances turned out to be really useful to the purpose. A voltammetric method for the determination of risperidone and olanzapine, two widely used atypical antipsychotics, has also been setup.

High-Performance Liquid Chromatographic Determination of Pantoprazole and Its Main Impurities in Pharmaceuticals

An RP-HPLC method for simultaneous separation and quantification of pantoprazole and its five main impurities in pharmaceutical formulations was developed and validated. The separation was accomplished on a Zorbax Eclipse XDB C18 column (5 microm particle size, 150 x 4.6 mm id) using a gradient with mobile phase A [buffer-acetonitrile (70 + 30, v/v)], and mobile phase B [buffer-acetonitrile (30 + 70, v/v)]. The buffer was 0.01 M ammonium acetate solution with addition of 1 mL triethylamine/L of the solution, adjusted to pH 4.5 with orthophosphoric acid. The eluent flow rate was 1 mL/min, the temperature of the column was 30 degrees C, and the eluate was monitored at 290 nm. Linearity (r = 0.999), recovery (97.6-105.8%), RSD (0.55-1.90%), and LOQ (0.099-1.48 microg/mL) were evaluated and found to be satisfactory. The proposed method can be used for simultaneous identification and quantification of the analyzed compounds in pharmaceutical formulations.

Recognition of tablet content by chemometric processing of differential scanning calorimetry curves—An acetaminophen example

Chemometric processing of differential scanning calorimetry (DSC) curves is rarely approached in the literature. This paper discusses and presents an example of building discriminant models against the presence of a compound in pharmaceutical formulations. The dataset containing 11 curves of pure substances and 21 signals of various OTC tablets was processed by partial least squares discriminant analysis (PLS-DA) against detection of these substances in the formulations. Good model was found in the case of acetaminophen (5 PLS factors included), due to good proportion between positive and negative training samples. The model was validated on external prediction set and was found to have following parameters: RMS equal to 0.1068 (98.8% of explained variance), RMSECV equal to 0.148 (97.7% of explained variance) and external predictive error (RMSEP) equal to 0.3918 (86.5% of explained variance).

Simultaneous chemiluminescence determination of amoxicillin and clavulanic acid using least squares support vector regression

In this work, both the batch and flow injection chemiluminescence (CL) methods have been proposed for the simultaneous determination of two structurally similar β-lactams including amoxicillin and clavulanic acid (CLAV). Chemiluminescence spectral overlap of these two structurally similar β-lactams is the main limitation for the simultaneous analysis of the two compounds. Least squares support vector regression (LS-SVR) was applied to relate concentration of both the compounds to their CL profiles. The parameters of the model, consisting of kernel parameter, σ 2, and the regularization parameter, γ, were optimized by constructing different LS-SVR models and the model with the minimum root mean squared error of cross-validation (RMSECV) for the calibration set was selected as the best model and its parameters were chosen as the optimized values. The performance of LS-SVR model was compared with Partial Least Squares (PLS) and the results revealed the superiority of the LS-SVR over PLS model. Under the optimized experimental conditions for both the compounds, when LS-SVR was applied, the detection limits obtained were 0.2 and 0.60 μmol L −1 in the batch mode and 0.3 and 0.5 μmol L −1 in the flow injection mode for CLAV and amoxicillin, respectively. The proposed method was utilized for the simultaneous determination of the compounds in pharmaceutical formulations and spiked plasma samples.