High-pressure Liquid Chromatographic Method Research Articles

Quechers Extraction Followed by HPLC Estimation of Steroidal Glycoalkaloids viz, α-Chaconine and α-Solanine in Different Solanaceae Family Vegetable Varieties

Glycoalkaloids are the alkaloid derived chemical constituents naturally produced in various plants species especially Solanaceae family. These chemical constituents protect the plant against predators as well as pathogens. The consumption of high dose of glycoalkaloids was toxic to humans. This study was purposeful to evaluate steroidal glycoalkaloids viz., α-chaconine and α-solanine quantity in different Solanaceae family vegetable varieties. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction using acetonitrile as extracting solvent was used for extracting steroidal glycoalkaloids in vegetable samples. In the optimized conditions, the standard chromatogram shows clear resolution of α-chaconine and α-solanine. The analytes were identified at 3.15 min for α-chaconine and 4.88 min for α-solanine. The optimized HPLC (High pressure liquid chromatography) method was validated. The method optimized using acetonitrile and 1.0 mM ammonium acetate buffer at pH 4.2 in 65:45 (v/v) as mobile phase at 0.6 mL/min, μBondapak C18 (10 μm, 4.6x250 mm) column as stationary phase and UV detection is selected at 218 nm. Findings suggest that this method was simple and convenient for the estimation of glycoalkaloids in vegetables. The glycoalkaloids was achieved under the acceptable levels of less than 200 mg/kg as prescribed by USFDA and German Federal Institute for Risk Assessment.

Effect of liposomal complexes of quercetin-rich flavonoids from French Marigold (Tagetes patula L.) on Jurkat cell viability

Background: Natural polyphenols are naturally used in traditional medicine to treat various diseases. Despite their healthful properties, ingesting phenolic compounds in food form does not provide a sufficient concentration for systemic therapeutic effects due to their low solubility in water, poor absorption, and fast metabolism. This problem has been solved by creating various composite pharmaceuticals from phenolic compounds using different methods to stabilize polyphenols. Objective: The purpose of this study was to assess the effects of the DPPA (1,2-palmitoyl phosphatidic acid) and DPPC (dipalmitoyl phosphatidylcholine) liposomes on the protective effects of a quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) on the viability of Jurkat cells. The study will examine both intact cells and cells that have been incubated under oxidative stress conditions. Materials and Methods: Quercetin-rich flavonoid fraction was extracted from a French Marigold (Tagetes patula L.) by thin-layer chromatography (TLC), High-pressure liquid chromatography (HPLC), and Liquid Chromatography-Mass Spectrometry (LC-MS) methods. Extract alone and in complex with dipalmitoyl phosphatidylcholine (DPPC) and 1,2- dipalmitoyl phosphatidic acid (DPPA) liposomes were added to the Jurkat cells culture at a rate of 2 mg/mL−1.The 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) test assayed Cell viability by evaluating cellular dehydrogenase activity. Results: Flavonoids were separated and identified in the marigold extracts by TLC, HPLC, and LC-MS methods. The spectrophotometric absorption spectrum of the quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) shows two peaks corresponding to benzoyl (254nm) and cinnamyl (375nm) aromatic rings. In the complex of quercetin-rich flavonoid fraction with DPPC and DPPA liposomes, the spectrophotometric absorption peak at 254nm was not detected, while the absorption intensity of the peak at 375nm was sharply reduced. The quercetin-rich flavonoid fraction alone and in combination with DPPC liposome increased intact and incubated under low- and high-intensity oxidative stress conditions Jurkat cells’ viability but did not reveal effect in combination with DPPA liposome. Conclusions: The quercetin-rich flavonoid fraction extracted from French Marigold (Tagetes patula L.) forms stable complexes with DPPC and DPPA liposomes that allow the storage of high content of phenolic compounds in lipid nanocapsules. The use of the liposomal system in the pharmaceutical and food industry allows for carried and controlled bioactive-compound release, which is considered one of the main strategies to improve and enhance the quality of food, providing preventative healthcare for the population and decreasing the risk of disease. Keywords: bioactive-compound, polyphenols, Quercetin, Liposomes, Jurkat cells, therapeutic effects, pharmaceuticals

HPLC method for the determination of thymoquinone in growth cell medium.

Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 μM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

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.

Simultaneous Quantification of Mometasone Furoate and Formoterol Fumarate in Bulk and Formulations by RP-HPLC Method

A simple and robust reversed-phase high pressure liquid chromatographic method was developed and validated for the quantification of Mometasone and Formoterol in bulk and fixed dosage forms. The column utilized for study was Inertsil C18, ODS was chosen for good peak shape. Ambient temperature was found to be suitable for the nature of drug solution. The mobile phase was streamlined with a ratio of 35:65 Methanol: Water was optimized for symmetrical peaks and good resolution. The flow rate was streamlined at 1.0 ml/min because of good peak area, satisfactory retention time and good resolution. Common λ-max was found to be at 278 nm and Injection volume was 20 µl which gave a good peak area. The percentage recovery was found to be 98.0-101.50 was linear and precise over the same range. Both system and method precision was found to be accurate and well within the acceptable limits. Detection limit was found to be 0.25 Mometasone and 0.34 for Formoterol. The analytical method was found linearity over the range of 20-80 ppm of the target concentration for both the drugs. The LOD and LOQ values for Mometasone, was observed to be 0.25 and 0.77 and for Formoterol was in the range of 0.34 and 1.05 consecutively. The analytical method passed both intermediate precision and robustness tests. On both cases, relative standard deviation was well satisfactory. The validation experiments met ICH standards. It inferred the method found to be simple, accurate, precise and linear. The method was found to be having suitable application in routine laboratory analysis with high degree of accuracy and precision.

Temperature-Sensitive Hydrogels as Carriers for Modulated Delivery of Acetaminophen.

The purposes of this study are the polymerization of temperature-sensitive copolymers based on N-isopropyl acrylamide and 10 mol % of 2-hydroxypropylmethacrylate, characterisations of their thermal, morphological and swelling properties, as well as the analysis of potential application in drug-delivery systems. Acetaminophen, the representative of non-steroidal anti-inflammatory drugs, was used as a model drug in this study. It is a common pain relief drug, which is also used for fever treatment. However, oral administration comes with certain health risks, mainly the overdose and frequent administration of up to four times a day. The goal of applying temperature-sensitive hydrogel is to enable extended administration once a day, depending on the body temperature. The swelling behavior of the obtained poly(N-isopropyl acrylamide-co-2-hydroxypropylmethacrylate) (p(NIPA/HPMA)) hydrogels and their temperature-sensitivity, kinetics and order of swelling processes at 18 and 38 °C were analyzed. The thermal properties of these hydrogels were observed by the DSC method, and the obtained thermograms showed both melting and glass transitions. The drug delivery system of p(NIPA/HPMA) hydrogels with loaded acetaminophen was analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy methods. Structural analysis of FTIR spectra indicates that non-covalent intermolecular interactions of the type of hydrogen bonds were formed among functional groups of acetaminophen and side-chains of p(NIPA/HPMA) hydrogels. The surface structure of p(NIPA/HPMA) hydrogels after drug loading indicates the acetaminophen presence into the pores of the hydrogel network, and their loading efficiency was higher than 92%. Qualitative and quantitative analysis of acetaminophen, determined by the high-pressure liquid chromatography method, showed that about 90-99% of the loaded amount was released from p(NIPA/HPMA) hydrogels within 24 h. Kinetic parameters of the acetaminophen release under simulated gastrointestinal conditions were determined. Based on obtained results, the drug delivery system of temperature-sensitive p(NIPA/HPMA) hydrogels with loaded acetaminophen could be suitable for additional investigation for modulated drug administration, e.g., for extended drug administration.

Analytical method validation on simultaneous estimation of Ozenoxacin and Benzoic acid in pharmaceutical formulation

AbstractIn this study, an accurate, simple, economical and precise Reversed-Phase High Pressure Liquid Chromatography (RP-HPLC) method was developed for the simultaneous estimation of Ozenoxacin and Benzoic Acid in a pharmaceutical cream formulation, according to the International Conference on Harmonisation (ICH) guidelines. Chromatographic separation was achieved by gradient elution, on RP-HPLC Instrument, equipped with column C8 (150 mm × 4.6 mm, 5 μm particle size) using Ultra Violet (UV) detector at 235 nm wavelength, by using Mobile Phase A: triethylamine, trifloroacetic acid and water (1:1:1000) and Mobile Phase B: methanol and Diluent: water, acetonitrile and triethylamine (500:500:1), at flow rate 0.8 mL min−1; injection volume of 20 μL; column oven temperature 45 °C and sample temperature: 25 °C; Run time: 15 min. All the validation parameters were within the acceptance criteria, as per ICH requirements, for Ozenoxacin and Benzoic acid. Consequently, this method has found to be validated, simple, rapid and successfully applicable, to the simultaneous estimation of Ozenoxacin and Benzoic acid by RP-HPLC, for routine analytical testing in quality control, with a run time of 15 min and for future research studies. Forced degradation of Ozenoxacin cream 1% w/w formulation was performed and found that validated method has stability indicating potential that needs to be further studied.

Long-Term Intake of Linezolid Elevates Drug Exposure and Reduces Drug Clearance and Elimination in Adults With Drug-Resistant Pulmonary Tuberculosis.

Pharmacokinetic (PK) studies are critical for dose optimization, and there is a paucity of linezolid (LZD) PK data for prolonged use in drug-resistant tuberculosis (DR-TB). Therefore, the authors evaluated the pharmacokinetics of LZD at two-time intervals in DR-TB during long-term use. PK evaluation of LZD was performed at the end of the 8th and 16th weeks of treatment in a randomly selected subset of adult pre-extensively drug-resistant pulmonary tuberculosis patients (n = 18) from a multicentric interventional study (Building Evidence to Advance Treatment of TB/BEAT study; CTRI/2019/01/017310), wherein a daily dose of 600 mg LZD was used for 24 weeks. Plasma LZD levels were measured using a validated high-pressure liquid chromatography (HPLC) method. The LZD median plasma C max was comparable between the 8th and 16th weeks [18.3 mg/L, interquartile range (IQR: 15.5-20.8 and 18.8 mg/L, IQR: 16.0-22.7, respectively)]. However, the trough concentration increased significantly in the 16th week (3.16 mg/L, IQR: 2.30-4.76), compared with the 8th week (1.98 mg/L, IQR: 0.93-2.75). Furthermore, compared with the 8th week, in the 16th week, there was a significant increase in drug exposure (AUC 0-24 = 184.2 mg*h/L, IQR: 156.4-215.8 versus 233.2 mg*h/L, IQR: 187.9-277.2), which corroborated with a longer elimination half-life (6.94 hours, IQR: 5.55-7.99 versus 8.47 hours, IQR:7.36-11.35) and decreased clearance (2.91 L/h, IQR: 2.45-3.33 versus 2.19 L/h, IQR: 1.49-2.78). Long-term daily intake of 600 mg LZD resulted in a significant elevation in trough concentration (>2.0 mg/L) in 83% of the study participants. Furthermore, increased LZD drug exposure may be partly because of decreased clearance and elimination. Overall, the PK data underscore the need for dose adjustment when LZDs are intended for long-term treatment.

Bioanalytical method development for quantification of trifluridine with its metabolites and tipiracil in spiked human plasma by solid-phase extraction (SPE) and HPLC with PDA detection

A novel high pressure liquid chromatography method coupled with diode array detector (HPLC-DAD) was developed and found to be efficient, sensitive, and selective for the simultaneous analysis of Trifluridine (TRI), and its metabolites 5-trifluoromethyl uracil (TFMU) and 5-carboxydeoxyuridine (CDMU) and Tipiracil (TIP) in a spiked human plasma. The HPLC-DAD technique that developed was validated in accordance with the most recent ICH M10 criteria. Sample pre-treatment was performed using a solid phase extraction with InertSep HLB reversed phase sorbent 30 mg/ml cartridge. The compounds were analyzed by liquid chromatography UV detection on a Inertsil C18 (250 mm × 4.6 mm ID, Particle size: 5 µm) with a mobile phase containing sodium acetate buffer pH (3.8; 10 mM) - acetonitrile (70:30, v/v) with flow rate of 0.8 ml/min with UV detection at 254 nm. The method was linear in the range 200–5000 ng/ml for TRI, TFMU, CDMU, and 100–1500 ng/ml for TIP. With the exception of the lower limit of quantification, where they were within ≤ 20%. The accuracy and precision for spiked human plasma were within ≤ 15% for all concentrations.

Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography.

Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products. Thereafter, the effects of the HA-containing eye products on corneal wound healing were investigated. The MW distributions and concentrations of HA in over the counter eye products were determined by size exclusion chromatography (SEC) high-pressure liquid chromatography (HPLC). The effects of the eye products containing HA on corneal wound healing were characterized both in vitro and in vivo using the scratch assay and the debridement wound model, respectively. The concentrations and MWs of HA were successfully determined within a range of 0.014 to 0.25 mg/mL using SEC HPLC. The concentrations of HA in the ophthalmic products varied from 0.14 to 4.0 mg/mL and the MWs varied from ∼100 kDa to >2500 kDa. All but one HA-containing eye product had an inhibitory effect on corneal wound healing, whereas pure HA promoted corneal wound healing. A novel SEC-HPLC method was developed for quantifying and characterizing the MW of HA in eye products. Although HA promoted corneal wound healing, HA-containing eye products inhibited corneal wound healing, likely caused by preservatives. SEC-HPLC could be implemented as a routine method for determining the form of HA in commercially available ophthalmic products.

Production of (10S,11S)-(-)-epi-Pyriculol and Its HPLC Quantification in Liquid Cultures of Pyricularia grisea, a Potential Mycoherbicide for the Control of Buffelgrass (Cenchrus ciliaris).

(10S,11S)-(-)-epi-pyriculol is a phytotoxic metabolite produced by Pyricularia grisea, a fungus identified as a foliar pathogen on the invasive weed species buffelgrass (Cenchrus ciliaris) in North America. The effective control of buffelgrass has not yet been achieved, and there is a need to develop effective and green solutions. Herbicides based on natural products and the use of phytopathogenic organisms could provide the most suitable tools for the control of weeds such as buffelgrass. Thus, one of the most relevant points to study about potential suitable phytotoxins such as (10S,11S)-(-)-epi-pyriculol is its production on a large scale, either by isolation from fungal fermentations or by synthesis. For these purposes, rapid and sensitive methods for the quantification of (10S,11S)-(-)-epi-pyriculol in complex mixtures are required. In this study, a high-pressure liquid chromatography (HPLC) method for its quantification was developed and applied to organic extracts from twelve P. grisea isolates obtained from diseased buffelgrass leaves and grown in potato dextrose broth (PDB) liquid cultures. The analysis proved that the production of (10S,11S)-(-)-epi-pyriculol is fungal-isolate dependent and strongly correlated with phytotoxic activity, shown by the P. grisea organic extracts in a buffelgrass radicle elongation test. The HPLC method reported herein allowed us to select the best strain for the production of (10S,11S)-(-)-epi-pyriculol and could be useful for selecting the best cultural conditions for its mass production, providing a tool for the use of this promising metabolite as a new bioherbicide for the control of buffelgrass.

Multivariate Analysis and Response Surface Modeling to Green Analytical Chemistry-Based RP-HPLC-PDA Method for Chromatographic Analysis of Vildagliptin and Remogliflozin Etabonate.

The fixed-dose combination (FDC) of vildagliptin (VDG) and remogliflozin etabonate (RGE) is used as antidiabetic medicine. Numerous reverse phase high-pressure liquid chromatographic (RP-HPLC) methods have been reported for the estimation of VDG and RGE using toxic organic solvents such as acetonitrile and methanol. These organic solvents are also hazardous to the environment. Hence, the robust and green analytical chemistry-based RP-HPLC-PDA method has been developed for chromatographic analysis of VDG and RGE for the safety of analysts and protection of the environment. The multivariate analysis has been carried out for the identification of critical method risk parameters (CMRPs) and critical method performance attributes (CMPAs) using principal component analysis (PCA). The identified CMRPs and CMPAs were linked with each other for optimization of the RP-HPLC-PDA method using DoE-based response surface modeling. The analytical design space (ADS) has been explored for robust chromatographic analysis of VDG and RGE. The chromatographic analysis of VDG and RGE has been carried out using Shim-Pack C18 column (250 mm L, 4.6 mm ID, 5.0 µm PS) and isopropyl alcohol-0.1% (v/v) formic acid (FA) in water (45 + 55, v/v, pH -3.5). The developed method has been validated in accordance with ICH Q2 (R1) guidelines. The method has been applied for the assay of VDG and RGE in their FDCs. The results of the assay were found in compliance with the labeled claims. The developed RP-HPLC-PDA method did not include any toxic or carcinogenic solvents. Hence, it is safe for analysts and the environment. The greenness profiles of the published and proposed RP-HPLC methods were evaluated by the national environmental method index (NEMI) scale, and the analytical greenness scores were calculated using the AGREE software. The developed method can be used as an eco-friendly tool in the pharmaceutical industry for routine analysis and quality control of FDCs of VDG and RGE. Development of a green and robust RP-HPLC method for the estimation of VDG and RGE using safe organic solvents. The analytical quality by design (AQbD) approach has been implemented in the development of a method to minimize solvent wastage. The method was applied for the assay of FDCs of VDG and RGE.

Biomedical Applications of Thermosensitive Hydrogels for Controlled/Modulated Piroxicam Delivery.

The objectives of this study are the synthesis of thermosensitive poly(N-isopropylacrylamide-co-2-hydroxypropyl methacrylate), p(NiPAm-HPMet), hydrogels and the analysis of a drug-delivery system based on piroxicam, as a model drug, and synthesized hydrogels. A high pressure liquid chromatography method has been used in order to determine both qualitative and quantitative amounts of unreacted monomers and crosslinkers from polymerized hydrogels. Swelling kinetics and the order of a swelling process of the hydrogels have been analyzed at 10 and 40 °C. The copolymers' thermal properties have been monitored by the differential scanning calorimetry (DSC) method. DSC termograms have shown that melting occurs in two temperature intervals (142.36-150.72 °C and 153.14-156.49 °C). A matrix system with incorporated piroxicam has been analyzed by using FTIR and SEM methods. Structural analysis has demonstrated that intermolecular non-covalent interactions have been built between side-groups of copolymer and loaded piroxicam. Morphology of p(NiPAm-HPMet) after drug incorporation indicates the piroxicam presence into the copolymer pores. Kinetic parameters of the piroxicam release from hydrogels at 37 °C and pH 7.4 indicate that the fluid transport mechanism corresponds to Fickian diffusion. As a result, formulation of thermosensitive p(NiPAm-HPMet) hydrogels with incorporated piroxicam could be of interest for further testing as a drug carrier for modulated and prolonged release, especially for topical administration.

Zearalenone (ZEN) and Its Metabolite Levels in Tissues of Wild Boar (Sus scrofa) from Southern Italy: A Pilot Study

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of the Fusarium genera, and is a contaminant of cereals and plant products. ZEN and its metabolites are considered endocrine disruptors, and could have various toxic effects on animals and humans. In recent years, there has been a significant demographic increase in wild boar (Sus scrofa) in many mountainous and hilly areas of Italy, including the Campania region, mainly due to global climate change. The wild boar can be defined as a generalist and omnivorous species capable of varying its diet; therefore, it can play a role as an environmental bioindicator towards contaminants such as mycotoxins. This study was conducted to evaluate, for the first time, the concentrations of ZEN and its metabolites in the liver, kidney, and muscle of 82 wild boars shot in their habitat by hunters with hunting permits in different localities of Avellino province (Campania region, Southern Italy) from 2021 to 2022. The samples were collected and analyzed with an SPE clean-up and high-pressure liquid chromatography method with fluorescence detection. The results indicated that ZEN and α-Zearalenol were present in most of the samples, suggesting that a plan to monitor these mycoestrogens is essential to achieve the goals of "One Health".

Newborn screening for sickle cell anemia in Antalya, Türkiye.

In a screening study conducted on adults, the prevalence of sickle cell traits in Antalya was found to be 0.24%. Since no screening studies have been conducted in the neonatal period in our region, the exact incidence has not been determined. In this study, we aim to report our experience of neonatal screening for sickle cell disease in Antalya, Türkiye. During a 14-month period, 2562 heel prick blood samples, taken on filter paper from Akdeniz University Hospital, Antalya Education and Research Hospital and Antalya Atatürk State Hospital and four other healthcare centers, were studied using the high pressure liquid chromatography method. Blood samples were studied using the `Sickle Cell Short Program` test method on a Bio Rad Variant device. In the study, no patients with sickle cell disease were identified. Four newborns who were sickle cell carriers (0.15%) and two newborns who were Hemoglobin D carriers (0.08 %), were found. Considering the efficiency and cost calculations made as a result of the data obtained from our study, it was concluded that sickle cell screening would not be effective in newborns. It seems more effective and economical to screen the children of parents, who are found to be at risk for Hemoglobin S carriage as a result of premarital tests.

Upravljanje resursima u HPLC - otkrivanje zelenog lica farmaceutske analize

High-pressure liquid chromatography (HPLC) is a technique of paramount importance in the analysis of pharmaceuticals because of its ability to separate moderately polar to less polar compounds, such as drugs and related substances.High-pressure liquid chromatography (HPLC) is a technique of paramount importance in the analysis of pharmaceuticals because of its ability to separate moderately polar to less polar compounds, such as drugs and related substances. The concept of green analytical chemistry (GAC) aims to provide more environmentally friendly and safer analytical methods in terms of reagents, energy, and waste. One of the major challenges of GAC is to find an appropriate approach to evaluate the greenness of analytical methods. An extension of GAC, called white analytical chemistry (WAC), has been introduced to consider not only environmental friendliness, but also other aspects that contribute to the sustainability of methods, such as analytical and economic or practical efficiency. HPLC methods are intrinsically not green, due to the high consumption of toxic organic solvents and the resulting generation of large amounts of toxic waste. Fortunately, there are many approaches to overcome the non-green character of HPLC methods. In this article, various modifications of the HPLC methods that increase its environmental friendliness are presented, as well as the various tools used to evaluate environmental friendliness. In addition, the new concept of white analytical chemistry is presented.

Hematological Parameters: Manual vs Automated Method; Among the β-Thalassemia and other Haemoglobinopathies in a Tertiary Care Hospital in Kolkata

Background: According to a report of WHO in 2007, 7% of world populations are carrier for Haemoglobindisorder and accurate and timely detection of various Hb variants including beta thalassemia trait can preventoccurrence of more serious disorders like thalassemia major in new-borns. But in a developing country like ususe of high pressure liquid chromatography (HPLC) is limited, manual testing is done and accurate assessmentis quite impossible. So, an observational cross-sectional study was done among 117 β-thalassemia and otherHemoglobinopathies cases and carriers.Objective: To find out the variation between the results of haematological tests obtained by automated countingchamber and manual method.Materials & Methods: This Observational, cross-sectional study was done at Thalassemia control unit and Physiologydepartment of R G Kar Medical College and Hospital, Kolkata. β-thalassemia and other Hemoglobinopathies casesand carriers detected by complete blood count with HPLC from Thalassemia Outpatient Department (OPD) andantenatal mothers from antenatal clinic (ANC) were the population of this study. Findings and variations of thefeatures of different hematological parameters amongst the β-thalassemia and other hemoglobinopathies werereviewed. A finding by HPLC was HbA, HbA2, HbF, HbD, and HbE.Results: Result of only Hb% obtained from HPLC and manual method was significantly different inβ- THALASSEMIA Trait (p=0.0001), HbE Trait (p=0.0001), HbS trait (p=0.0001) and not significant in HbE diseaseand no other Red cell indices were significantly different in two methods. Conclusion: As hematological featureslike TC of RBC or PCV results by manual method shows no significant differences with HPLC/Automated counter assessment, such methods can be utilized even at primary level cost effectively for assessment of hematologicaldisorders. But significant difference is seen in estimating Hemoglobin percentage in between Shalli‘s method andHPLC/Automated counter assessment. So some alternate method may introduce at lower level of health systemother than Shalli’s method

Validation of the HPLC Analytical Method for the Determination of Chemical and Radiochemical Purity of Ga-68-DOTATATE.

Ga-68-DOTA-peptides targeting somatostatin receptors have been assessed as a valuable tool in neuroendocrine tumors imaging using positron emission tomography (PET). A new selective and sensitive high-pressure liquid chromatography (HPLC) method was developed for determining chemical and radiochemical purity of Ga-68-DOTATATE (PET) tracer. The identification of peaks was achieved on a symmetry C18 column 3 μm 120Å (3.0 mm × 150 mm spherical particles) using (A) water + 0.1% trifluoroacetic acid (TFA) and (B) acetonitrile + 0.1% TFA, as the mobile phases at a flow rate of 0.600 mL/min and monitored at 220 nm. The run time was 16 min. The method was validated to fulfill International Conference on Harmonization requirements and EDQM guidelines, and it included specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision. The calibration curve was linear over the concentration range from 0.5 to 3 μg/ml, with a correlation coefficient (r2) equal to 0.999, average coefficient of variation (CV%) 2%, and average bias% did not deviate more than 5% for all concentrations. The LOD and LOQ for DOTATATE were 0.5 and 0.1 μg/mL, respectively. The method was considered precise, obtaining coefficients of variation between 0.22% and 0.52% for intraday and 0.20% and 0.61% for interday precision. Accuracy of method was confirmed with average bias% that did not deviate more than 5% for all concentrations. All results were acceptable and this confirmed that the method is suitable for its intended use in routine quality control of Ga-68-DOTATATE to guarantee the high quality of the finished product before release.

HPLC Method Validation for the Estimation of Lignocaine HCl, Ketoprofen and Hydrocortisone: Greenness Analysis Using AGREE Score.

In the current study, the reversed-phased high-pressure liquid chromatography (RP-HPLC) method was proposed for the estimation of lignocaine hydrochloride (LIG), hydrocortisone (HYD) and Ketoprofen (KET) according to International Conference for Harmonization (ICH) Q2 R1 guidelines, in a gel formulation. The chromatographic evaluation was executed using Shimadzu RP-HPLC, equipped with a C8 column and detected using UV at 254 nm wavelength, using acetonitrile and buffer (50:50) as a mobile phase and diluent, at flow rate 1 mL/min and n injection volume of 20 μL. The retention time for LIG, HYD, and KET were 1.54, 2.57, and 5.78 min, correspondingly. The resultant values of analytical recovery demonstrate accuracy and precision of the method and was found specific in identification of the drugs from dosage form and marketed products. The limit of detection (LOD) for LIG, HYD, and KET were calculated to be 0.563, 0.611, and 0.669 ppm, while the limit of quantification (LOQ) was estimated almost at 1.690, 1.833, and 0.223 ppm, respectively. The AGREE software was utilized to evaluate the greenness score of the proposed method, and it was found greener in score (0.76). This study concluded that the proposed method was simple, accurate, precise, robust, economical, reproducible, and suitable for the estimation of drugs in transdermal gels.

Cost-Effective HPLC Method for Identification of Rosmarinic Acid in Methanol Extract of Ocimum Basilicum

Prescribing herbal remedies is now becoming common by many practitioners, who propose herbal treatments along with conventional medicine systems to treat various ailments considering their synergistic effects. Rosmarinic acid is a potent phenolic compound and caffeic acid derivative. It has many miraculous biological activities. Anti-allergic, anti-angiogenic, anti-depressant, anti-inflammatory, antitumor, anti-microbial activity, and antiviral effect of rosmarinic acid has already been documented in previous literature. It is widely present in many formulations because of its hepatoprotective and antioxidant effect. This study aimed to develop and validate a RP-HPLC method for the standardization of O. basilicum L. raw material and extracts by using rosmarinic acid as a marker. The study also aimed to quantify the rosmarinic acid content in the methanol extract of O. basilicum leaves. A simple and cost-effective High Pressure Liquid Chromatography (HPLC) method for determining rosmarinic acid in Ocimum basilicum plant extract has been developed and validated. Methanol-water-orthophosphoric acid in the ratio of 95:5:1 was used as the mobile phase using a C18 reversed-phase column for the HPLC method. Signals were detected at 254 nm. The rosmarinic acid content was found to be 4.2 mg/10 mg of the plant extract. The method is sensitive and reproducible and ideal for quick routine analysis and can be employed for detecting rosmarinic acid in other herbs. Despite the availability of literature on the medicinal properties of this plant and its chemical constituents, only a limited number of papers have been published on the determination of the rosmarinic acid in Ocimum basilicum plant using RP-HPLC. Moreover, this is the first report that quantifies rosmarinic acid in the plant extract by HPLC without using expensive columns and complex buffer systems that deteriorate the chromatographic columns and in turn, enhance the cost of the used process.