Reversed-phase Liquid Chromatographic Method Research Articles

Sensitive and validated UPLC-MS/MS method for the identification and characterization of forced degradation products for felodipine

AbstractA rapid, stability indicating reverse phase liquid chromatographic method was developed for the determination of purity of Felodipine in active pharmaceutical substance form in the presence of its impurity and its degradation products. To develop the method which is also compatible to liquid chromatographic mass spectroscopic technique. The developed method is also used to determine the assay of Felodipine in bulk drug form. The drug is subjected to various stress conditions like acidic, basic, oxidation, UV light and thermal conditions. Considerable degradation was observed during base hydrolysis. Two degradation products were identified. The Waters Acquity UPLC BEH C18, 2.1 × 100 mm, 1.7 µm Column was used to achieve chromatographic separation. The gradient conditions, diluent and injection volume were optimized to achieve the acceptable resolution between impurities and its degradation products from Felodipine and to get good peak shapes. The masses were determined for main compound and its identified degradation products. Further, the characterization studies for main compound and its degradation products were performed using LCMSMS Q-TOF.

Stability-Indicating UPLC-PDA-QDa Methodology for Carvedilol and Felodipine in Fixed-Dose Combinations Using AQbD Principles

The development of analytical procedures, in line with the recent regulatory requirements ICH Q2 (R2) and ICH Q14, is progressing, and it must be able to manage the entire life cycle of the methodology. This is also applicable to and especially challenging for combinations of drug substances and dosage form. A reliable and efficient, stability-indicating, MS-compatible, reverse-phase ultra-performance liquid chromatographic (UPLC®) method was developed for the determination of carvedilol and felodipine in a combination oral dosage form. The development of the method, performed using analytical quality by design (AQbD) principles, was in line with the future regulatory requirements. Furthermore, the fixed-dose combination dosage forms are a clear solution to the polypharmacy phenomenon in the elderly population. The main factors evaluated were the mobile phase buffer, organic modifier, column, flow, and column temperature. The optimum conditions were achieved with a Waters Acquity HSS T3 (100 × 2.1 mm i.d., 1.8 µm) column at 38 °C, using ammonium acetate buffer (5 mM, pH 4.5) (Solution A) and MeOH (Solution B) as mobile phases in gradient elution (t = 0 min, 10% B; t = 1.5 min, 10% B; t = 12.0 min, 90% B; t = 13.0 min, 10% B; t = 15.5 min, 10% B) at a flow rate of 0.2 mL/min and UV Detection of 240 and 362 nm for carvedilol (CAV) and felodipine (FLP), respectively. The linearity was demonstrated over concentration ranges of 30–650 µg/mL (R2 = 0.9984) (CAV) and 32–260 µg/mL (R2 = 0.9996) (FLP). Forced degradation studies were performed by subjecting the samples to hydrolytic (acid and base), oxidative, and thermal stress conditions. Standard solution stability was also performed. The proposed validated method was successfully used for the quantitative analysis of bulk, stability, and fixed-dose combination dosage form samples of the desired drug product. Using the AQbD principles, it is possible to generate methodologies with improved knowledge, leading to high-quality data, lower operation costs, and minimum regulatory risk. Furthermore, this work paves the way for providing a platform of robust analytical methods for the simultaneous quantification of innovative on-demand new dose combinations.

Comparison of Classical and Green RPLC Methods in the Determination of pKa Values of Some Piperazine Group Antihistamines.

In this study, protonation constant values and liquid chromatographic behaviors of hydrophobic cyclizine, chlorcyclizine, hydroxyzine, cinnarizine, cetirizine, meclizine, and buclizine in some water-organic solvent binary mixtures were examined for the first time using classical and green reverse phase liquid chromatography methods. In the isocratic study, the relationship of the retention time and mobile phase pH in water-organic solvent binary mixtures containing acetonitrile (45, 50, 55, 60, 65, v/v), methanol (60, 65, 70, 75, v/v) and ethanol (45, 50, 55, 56, 59, 60, 62, 65, v/v) were determined at 37 °C. In the study, XBridge C18 and Gemini NX C18 columns suitable for the chemical properties of basic compounds were used. The obtained liquid chromatographic data were analyzed using the linear solvation energy relationship methodology and the SOLVER program. The aqueous protonation constant values of the investigated compounds were calculated using the linear relationship between the protonation constant data calculated in studied binary mixtures and some macroscopic constant values of the solvents used. The greenness of methods developed using three different solvents was evaluated with the Analytical Greenness Metric Approach, the Green Analytical Procedures Index, and the Green Solvent Selection Tool approaches.

Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA

Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. Column temperature, mobile phase flow rate and ion-pair selection were each studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100 mM Triethylamine, enabled the highest resolution separation for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the formed degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well monitored, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.

Label-Free Composition Analysis of Supramolecular Polymer-Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector.

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving the desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel composition is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e., proteins) and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with (1) a multi-angle light scattering detector (RPLC-MALS) or (2) high resolution mass spectrometry (RPLC-MS) and a Fourier-transform based deconvolution algorithm. We envision that this analytical strategy could be generalized to characterize critical quality attributes of other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

Advances in the Separation and Detection of Secondary Organic Aerosol Produced by Decamethylcyclopentasiloxane (D5) in Laboratory-Generated and Ambient Aerosol.

Decamethylcyclopentasiloxane (D5), a common ingredient in many personal care products (PCPs), undergoes oxidation in the atmosphere, leading to the formation of secondary organic aerosol (SOA). Yet, the specific contributions of D5-derived SOA on ambient fine particulate matter (PM2.5) have not been characterized. This study addresses this knowledge gap by introducing a new analytical method to advance the molecular characterization of oxidized D5 and its detection in ambient aerosol. The newly developed reversed phase liquid chromatography method, in conjunction with high-resolution mass spectrometry, separates and detects D5 oxidation products, enabling new insights into their molecular and isomeric composition. Application of this method to laboratory-generated SOA and urban PM2.5 in New York City expands the number of D5 oxidation products observed in ambient aerosol and informs a list of molecular candidates to track D5-derived SOA in the atmosphere. An oxidation series was observed in which one or more methyl groups in D5 (C10H30O5Si5) is replaced by a hydroxyl group, which indicates the presence of multistep oxidation products in ambient PM2.5. Because of their specificity to PCPs and demonstrated detectability in ambient PM2.5, several oxidation products are proposed as molecular tracers for D5-derived SOA and may prove useful in assessing the impact of PCPs-derived SOA in the atmosphere.

Mechanistic studies to understand peak tailing due to sulfinic acid- and carboxylic acid-silanophilic interactions in reversed-phase liquid chromatography

Silanophilic interactions are a primary contributor to peak tailing of acidic pharmaceutical compounds, thus a thorough understanding is especially important for reversed-phase liquid chromatography (RPLC) method development. Herein, a sulfinic acid compound that exhibited severe peak tailing in RPLC with acidic mobile phases was carefully studied. Results indicated that the neutral protonated form of the sulfinic acid is involved in the strong interaction that leads to peak tailing, but that tailing can be mitigated with a blocking effect achieved through use of acetic acid modifier in the mobile phase. Peak tailing was also observed with other structurally-similar sulfinic acids and carboxylic acids but was, in general, less severe with the latter. The Hydrophobic Subtraction Model (HSM) was applied to six commercial C18 columns that exhibited different tailing behaviors for the sulfinic acid compound in attempts to identify key sites of interaction within the stationary phase. A combination of heated acid column wash experiments and density functional theory (DFT) calculations indicate that the differential interactions of the acids with vicinal silanol pairs in the stationary phase play a major role in peak tailing.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Reverse phase liquid chromatographic method for the measurement of uranium in process stream solutions from uranium extraction facility

Abstract The present paper describes a simple method for the determination of uranium in uranium process stream solutions by reversed-phase liquid chromatographic method using C18 column, mixture of α-hydroxy isobutyric acid and methanol as eluent and flow-through spectrophotometer detector. This method has an advantage that interfering elements like rare-earth elements do not retain on the column and elute near the solvent peak thereby not interfering with uranium elution peak. Different eluents and parameters like pH, concentration of eluent are studied for the purpose. The spiking standard recovery was found to be more than 90 %. The developed method has been compared with other techniques and found to be in agreement.

Origanum majorana L. polyphenols: in vivo antiepileptic effect, in silico evaluation of their bioavailability, and interaction with the NMDA receptor.

Introduction: Epilepsy is a chronic brain disease characterized by repeated seizures and caused by excessive glutamate receptor activation. Many plants are traditionally used in the treatment of this disease. This study aimed to evaluate the bioavailability of a polyphenolic extract obtained from Origanum majorana L. (OMP) leaves, as well as its antiepileptic activity and its potential mechanism of action. Methods: We have developed and validated a simple, rapid, and accurate stability-indicating reversed-phase liquid chromatographic method for the simultaneous determination of caffeine and quercetin in rat plasma. The OMP antiepileptic effect was evaluated with pilocarpine-induced seizures, and a docking method was used to determine the possible interaction between caffeic acid and quercetin with the N-methyl-D-aspartate (NMDA) receptor. Results and Discussion: Both compounds tested showed low bioavailability in unchanged form. However, the tested extract showed an anticonvulsant effect due to the considerably delayed onset of seizures in the pilocarpine model at a dose of 100mg/kg. The molecular docking proved a high-affinity interaction between the caffeic acid and quercetin with the NMDA receptor. Taken together, OLP polyphenols demonstrated good antiepileptic activity, probably due to the interaction of quercetin, caffeic acid, or their metabolites with the NMDA receptor.

Reverse Phase Liquid Chromatography Method Optimization for Analysis of Some Azole Group Antifungal Compounds

Bu çalışmada azol türevi antifungal ilaç etken maddeleri klotrimazol, lanokonazol ve klimbazolün ters faz sıvı kromatografi (RPLC) yöntemi kullanılarak geliştirilen metot optimizasyonunda, bileşiklerin kapasite faktörü değerleri ve analizlendikleri hareketli fazdaki metanol derişimi ilişkisi esas alınmıştır. İyonlaşan gruba sahip bu bileşiklerin optimizasyonunda hareketli faz pH değeride optimizasyonda bir diğer etkendir. İzokratik bir ayırma için kapasite faktörü değerlerinin 1-5 aralığında olması tercih edilmektedir. RPLC yöntemiyle gerçekleştirilen klotrimazol, lanokonazol ve klimbazol bileşiklerinin aynı anda tayini için yapılan optimizasyonda %50, %55 ve %60 (v/v) metanol içeren metanol-su ikili karışımları kullanılmıştır. Ayırma için çalışılan ortamlarda iki hareketli faz pH değerindeki (pH 4,0 ve 5,5) alıkonma zamanları belirlenmiştir. Hidrofobik özellikteki bileşiklerin tayini için Zorbax SB-CN (4,6x150mm) kolon tercih edilmiştir. Kolon sıcaklığı 37oC’de ve akış hızı da 1 mL/dakikada sabitlenmiştir. Optimizasyon koşullarının belirlenmesinde logk- % (v/v) metanol ilişkisi ve bileşiklerin ayrılmalarını gösteren seçicilik faktörü (α) parametresinden yararlanılmıştır.

Characterization of Dye-Loaded Poly(lactic-co-glycolic acid) Nanoparticles by Comprehensive Two-Dimensional Liquid Chromatography Combining Hydrodynamic and Reversed-Phase Liquid Chromatography.

Analytical methods for the assessment of drug-delivery systems (DDSs) are commonly suitable for characterizing individual DDS properties, but do not allow determination of several properties simultaneously. A comprehensive online two-dimensional liquid chromatography (LC × LC) system was developed that is aimed to be capable of characterizing both nanoparticle size and encapsulated cargo over the particle size distribution of a DDS by using one integrated method. Polymeric nanoparticles (NPs) with encapsulated hydrophobic dyes were used as model DDSs. Hydrodynamic chromatography (HDC) was used in the first dimension to separate the intact NPs and to determine the particle size distribution. Fractions from the first dimension were taken comprehensively and disassembled online by the addition of an organic solvent, thereby releasing the encapsulated cargo. Reversed-phase liquid chromatography (RPLC) was used as a second dimension to separate the released dyes. Conditions were optimized to ensure the complete disassembly of the NPs and the dissolution of the dyes during the solvent modulation step. Subsequently, stationary-phase-assisted modulation (SPAM) was applied for trapping and preconcentration of the analytes, thereby minimizing the risk of analyte precipitation or breakthrough. The developed HDC × RPLC method allows for the characterization of encapsulated cargo as a function of intact nanoparticle size and shows potential for the analysis of API stability.

Determination of isohydrafural and methyluracil in ear drops by high performance liquid chromatography

Abstract Objective The aim of current study was the development and validation of a reversed-phase high-pressure liquid chromatography (RPHPLC) method for the quantitative determination of two active ingredients, isohydrafural (IHF) and methyluracil (MU) in fixed dose combination ear drops. Methods: An efficient separation of the two compounds was achieved on a Teknokroma C1 5μm (150*4,6mm) column, with a methanol: water 60:40 (V: V) mobile phase, at 300C temperature and 0.6 mL/min flow-rate. The total analysis time was 5.5 min. Result: The verified validation parameters were: linearity, selectivity, specificity, precision (repeatability and reproducibility), robustness, limit of detection and quantification. Conclusions: Good separation and lack of interference from other chromatographic peaks, rapidanalysis times were obtained. The newly developed chromatographic method can be applied for the analysis of fixed dose combination ear drops with isohydrafural and methyluracil.

A Rapid, Simplified and Validated Reverse Phase Liquid Chromatography Method for Quantitation of Molnupiravir and Its Generic Versions

Molnupiravir, an orally active RdRp inhibitor, though originally developed to use against influenza, has become a promising therapeutic candidate for COVID-19 infection by acting as prodrug of the nucleoside analog b-d-N4-hydroxycytidine (NHC or EIDD- 931) in the isopropyl ester form. Accordingly, it has been approved by MHRA, UK in November 2021 to use against COVID-19. This work presents the development and validation of a high-performance liquid chromatography (HPLC) method for the rapid and accurate assessment of molnupiravir. The objective of this approach is to ensure the quality control of Lagevrio® capsules and their generic versions available in Bangladesh. The separation was carried out by isocratic elution through C18 column (4.6 mm × 250 mm i.d., 5 µ particle size) at 40 °C temperature at a constant flow rate of 1.0 mL/min using acetonitrile and 1% orthophosphoric acid as buffer (20:80, v/v). A 10 mL sample was injected and elution was monitored at 210 nm wavelength. The calibration curve over the concentration range of 80-120% were found to be linear (r2 = 0.9998), the %RSD for intra-day (0.31%) and inter-day (0.73%) precision indicate good precision where %recovery was found in the acceptable range of 99.9-100.4% as per ICH guidelines. The method was successfully applied to commercial pharmaceuticals, Lagevrio® and its generic versions and and the results were found to be consistent with the label claims (95-105%).

Use of green analysis methods to determine the ionization constant values of isoxazolyl penicillins

In this study, an environmentally friendly analysis of penicillinase-resistant antibiotics oxacillin, dicloxacillin, and cloxacillin was carried out by liquid chromatography methods using ethanol solvent that is not harmful to human health and the environment. Ionization constant (wwpKa) values of isoxazolyl penicillins in ethanol–water binary mixtures containing 20–35% (v/v) ethanol at 37 °C were obtained by green reverse phase liquid chromatography and micellar liquid chromatography methods known as green chromatography. The developed methods are based on a model that relates the chromatographic behavior of the identified analytes to pH. The linear relationship between the microscopic solvatochromic parameter ETN and the retention values of the compounds was determined depending on the change of organic solvent in the mobile phase. Using the obtained chromatographic data, conditions that ensure the simultaneous separation of the compounds for both methods were determined. The relationship between the calculated sspKa values and the macroscopic properties of ethanol was investigated. The agreement between the pKa (wwpKa) values in the water medium determined by this approach was determined by t-test at a 95% confidence level for both methods. The wwpKa values of the compounds at 25 °C were also estimated using Abraham solute parameters without any experimental work.

Glycerol-Based Green RP-HPLC Method for Simultaneous Determination of Methionine and Paracetamol in Pharmaceutical Tablets

Methionine and paracetamol are combined in dosage forms due to the ability of methionine to compensate the deficiency in glutathione in hepatic patients. In this work, an innovative simple time-saving green RP-HPLC method using glycerol as a solely green mobile phase with water for the first time was developed for the simultaneous determination of methionine and paracetamol in their standard and in Hepamol® tablets. The chromatographic conditions were optimized using factorial design with the aid of Minitab 17® Software. The method was performed on a C18 column at 38 °C, and a mobile phase consisting of glycerol and phosphate buffer (pH 2.4) (40:60, v/v), using diode array detector at 210 nm. The mixture was separated in 5 min. The developed method was validated in accordance with ICH requirement over linearity ranges of 10–90 μg/mL for both drugs, and LODs were 3.33 μg/mL for both methionine and paracetamol. Glycerol has high safety, low UV cut-off point, low flammability, and its viscosity can be manipulated when diluted with water. The procedure was compared to the reported reversed phase liquid chromatography method in the terms of their greenness with the green analytical procedure index and the analytical eco-scale. This work breaks new ground for scientists to use glycerol in greener RP-HPLC applications.

Development and validation of reverse - phase HPLC method for detection of tetracycline residue in fresh milk samples

Dairy products are highly required by humans for growth, development and repairs as they are good sources of essential nutrients. Indiscriminate administration of veterinary drugs for prophylactic and therapeutic purposes could lead to the presence of their residues in dairy products such as milk. Reversed-phase high performance liquid chromatographic method with UV detection was developed and validated for detection of tetracycline in milk from five different samples. Mobile phase consisting of a mixture of deionized water, acetonitrile, monobasic potassium and acetic acid was delivered at a flow rate of 1.0 mL/min. The column used was Agilent ZORBAX ODS 4.6 X 150 mm 5μm.. The eluent was monitored by UV detection at 220 nm. Analysis was performed at 35oC and the total run time was 20 minutes. Analytical parameters such as linearity, accuracy, precision and limit of detection were determined by validation procedure and found to be satisfactory. Calibration curve was linear over the concentration range of 0.01- 0.5 mg/mL with an R2 of 0.9978. The limits of detection and limit of quantification were found to be 0.00023 mg/mL and 0.00110 mg/mL, respectively. The % RSD is found to be 3.62, 0.23 and 0.44 for 0.01, 0.1 and 0.5 mg/mL concentration with percentage recovery of 78.7 – 98.6 %. Generally, the developed method was found to be simple, rapid, precise and accurate for quality control of tetracycline in fresh milk samples.

Changes in the cis-trans isomer selectivity of a reversed-phase liquid chromatography column during use with acidic mobile phase conditions

Reversed-phase liquid chromatography (RPLC) is the analytical tool of choice for monitoring process-related organic impurities and degradants in pharmaceutical materials. Its popularity is due to its general ease-of-use, high performance, and reproducibility in most cases, all of which have improved as the technique has matured over the past few decades. Nevertheless, in our work we still occasionally observe situations where RPLC methods are not as robust as we would like them to be in practice due to variations in stationary phase chemistry between manufactured batches (i.e., lot-to-lot variability), and changes in stationary phase chemistry over time. Over the last three decades several models of RPLC selectivity have been developed and used to quantify and rationalize the effects of numerous parameters (e.g., effect of bonded phase density) on separation selectivity. The Hydrophobic Subtraction Model (HSM) of RPLC selectivity has been used extensively for these purposes; currently the publicly available database of column parameters contains data for 750 columns. In this work we explored the possibility that the HSM could be used to better understand the chemical basis of observed differences in stationary phase selectivity when they occur – for example, lot-to-lot variations or changes in selectivity during column use. We focused our attention on differences and changes in the observed selectivity for a pair of cis-trans isomers of a pharmaceutical intermediate. Although this is admittedly a challenging case, we find that the observed changes in selectivity are not strongly correlated with HSM column parameters, suggesting that there is a gap in the information provided by the HSM with respect to cis-trans isomer selectivity specifically. Further work with additional probe molecules showed that larger changes in cis-trans isomer selectivity were observed for pairs of molecules with greater molecular complexity, compared to the selectivity changes observed for simpler molecules. These results do not provide definitive answers to questions about the chemical basis of changes in stationary phase chemistry that lead to observed differences in cis-trans isomer selectivity. However, the results do provide important insights about the critical importance of molecular complexity when choosing probe compounds and indicate opportunities to develop improved selectivity models with increased sensitivity for cis-trans isomer selectivity.

Profiling of regioisomeric triacylglycerols in pistachio nuts by high-performance liquid chromatography-electrospray ionization mass spectrometry

In this contribution, a non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-MS) is used for the identification of triacylglycerol (TAG) down to the regioisomersíc level in pistachio lipids. Furthermore, for effective profiling of TAG, a two-dimensional (2D) map was employed to identify the entire homologous series of TAGs especially, odd-numbered fatty acid and long-chain derivatives. Notably, TAGs were observed as pairs of regioisomers with distinct retention times and tandem mass spectra. Furthermore, more than 100 TAGs containing 26 fatty acids with 51-63 carbon atoms and 0-3 double bonds were identified in different pistachio samples. The developed method is effective and facile for the fast quantification of complex TAGs in pistachio samples at high selectivity.

Stability-indicating method development and validation for quantitative estimation of organic impurities of the antidiabetic drug glipizide in drug substance and pharmaceutical dosage form using HPLC.

Glipizide is an antidiabetic drug used for the treatment of type 2 diabetes. A simple, reliable and robust reverse-phase liquid chromatographic method (RP-HPLC) was developed and validated as per International Conference on Harmonization Q2(R1) for estimating the impurities of glipizide in pharmaceutical formulations. The chromatographic separation was carried out on a Phenomenex Luna C18 (2), 250 × 4.6mm, 5μm with a binary solvent delivery system [MP-A, a homogenous mixture of water and acetonitrile in a ratio of 90:10 (v/v) and 1ml of orthophosphoric acid; and MP-B, a homogenous mixture of water and acetonitrile in a ratio of 10:90 (v/v) and 1ml of orthophosphoric acid] with a detection wavelength of 225 nm, a column temperature of 30°C, a flow rate of 1.5ml/min, and an injection volume of 20 μl. All process, degradant and unknown impurities were separated well with a resolution >2.2 and were estimated accurately without any interference. The recovered values and regression values were 98.7-100.5% and R2 > 0.9999, respectively. The recovery and linearity studies covered the quantitation limit to 150% of the specification limit. The stability-indicating properties of the developed RP-HPLC method was assessed from the forced degradation studies. The developed method was successfully applied for real-time sample analysis of the glipizide dosage form.