Normal-phase High-performance Liquid Research Articles

The Development of One New Normal Phase Liquid Chromatography Method and Thermodynamic Investigation of Olodaterol Hydrochloride Enantiomer.

In order to improve and replace the enantiomer method outlined in the olodaterol hydrochloride draft monograph (From the European Pharmacopoeia forum), one new, simple, and fast enantioselective normal phase high-performance liquid chromatography chiral method was developed on polysaccharide-based Chiral MX (2) (4.6 × 250 mm, 5 μm) column. n-Hexane, ethanol, and diethylamine in the ratio of 40:60:0.1 (V/V/V) were selected as mobile phase at a flow rate of 0.8 mL/min, and the detection was performed on a photodiode array detector at 225 nm with 5 μL injection volume. The column temperature was set at 40°C for better peak shape and sensitivity. The analysis time can be shortened to 15 min, whereas the resolution between enantiomer and olodaterol was found to be even more than 10.0, which was far better than that obtained with the reported method in this draft monograph. The developed chiral method was validated in accordance with ICH Q2 (R1), including specificity, LOD&LOQ, precision, linearity, accuracy, and robustness. Thereby, the proposed method was demonstrated to be suitable for the determination of enantiomer inolodaterol hydrochloride bulk drug and drug product. Besides, the thermodynamic parameters were evaluated on the basis of Van't Hoff plots that was used to explain correlative chiral recognition mechanisms with the chiral stationary phase.

Improving the Current European Pharmacopoeia Enantio-Selective HPLC Method for the Determination of Enantiomeric Purity in Atorvastatin Calcium Salt Drug Substance

Atorvastatin (ATV) is a well-established lipid-lowering agent. ATV has two stereogenic centers, and of the four possible stereoisomers, only the (3R,5R) form is used therapeutically. The European Pharmacopoeia (EP) monograph 2022 for ATV calcium salt describes a normal-phase high-performance liquid chromatography (HPLC) method for the determination of enantiomeric purity in both drug substance and working standard samples, based on a 150 mm × 4.6 mm Chiralpak AD-H column. The main problems with this method are the very long analysis time and the high solvent consumption. Here, an alternative chromatographic protocol was developed using the Chiralpak AD-3 column (250 mm × 4.6 mm) packed with 3 μm silica particles instead of the 5 μm silica particles of the Chiralpak AD-H chiral stationary phase and characterized by the same polysaccharide selector, amylose-tris(3,5-dimethylphenylcarbamate). Using a mobile phase consisting of a mixture of n-hexane-ethanol-formic acid 90:10:0.1 (v/v/v) as the mobile phase and setting the flow rate and column temperature to 1.0 mL min−1 and 35 °C, respectively, a simultaneous stereo-selective separation was achieved within 35 min without observing any overlap between the enantiomeric impurity peak and peaks related to other ATV impurities. Compared to HPLC EP conditions, the analysis time to elute all the potentially related substances was faster and significantly less mobile phase volume was required. The linearity of the method has been demonstrated in the range of 4.4 μg mL−1 to 1000 μg mL−1 (R2 > 0.999). At a concentration of 4.4 μg mL−1, which is 0.075% of the test solution (5.8 mg mL−1, as ATV free acid), the signal-to-noise ratio was found to be 20.

Retention mechanisms of amitriptyline and its impurities on amide, amino, diol, and silica columns in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has been widely applied to challenging analysis in biomedical and pharmaceutical fields, bridging the gap between normal-phase high-performance liquid chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). This paper comprehensively explores the retention mechanisms of amitriptyline and its impurities A, B, C, D, F, and G on amide, amino, diol, and silica columns. Dual HILIC/RP-HPLC retention mechanisms were developed, and transitional points between HILIC and RP-HPLC mechanisms were calculated on amide, diol, and silica columns. Adsorption and partition contributions to overall retention mechanisms were evaluated using Python software in HILIC and RP-HPLC regions. The cation exchange mechanism dominates overall retention for ionized analytes in the silica column (R2>0.995), whereas the retention of ionized analytes increases with pH. Impacts of acetonitrile content, buffer ionic strength, and pH, along with their interactions on the retention of ionized analytes in the silica column, were determined using the chemometric approach. Acetonitrile content showed the most significant impact on the retention mechanisms. These findings highlight that a detailed investigation into retention mechanisms provides notable insights into factors influencing analyte retention and separation, promising valuable guidance for future analysis.

Chiral LC-PDA-ORD Method for The Separation of Linagliptin Enantiomers On Coated Polysaccharide Based Amylose Tris (3, 5-Dimethylphenylcarbamate) Stationary Phases

Chiral normal phase high performance liquid chromatographic (chiral-HPLC) was designed and verified for the separation of linagliptin enantiomers using coated polysaccharide chiral stationary phases. The stationary phase was amylose tris (3, 5-dimethylphenylcarbamate) (250x4.6mm, 5 µm), while the mobile phase was a mixture of 50:50:0.1% v/v. With a flow rate of 1 mL/min, orthophosphoric acid was mixed with hexane, isopropyl alcohol, and diethyl amine to achieve a pH of 5.2. The detection was seen at 225 nm. The optical rotatory dispersion (ORD) polarimeter was connected in series to the PDA outlet in order to determine the enantiomer conformation. The linagliptin retention times were found to be 5.454 and 8.772 minutes. Between 3.9 and 23.4 µg/ml, enantiomers were discovered to be linear, with a correlation coefficient of 0.9995. This method was validated in terms of linearity, LOD, LOQ, precision, accuracy, and robustness studies in accordance with ICH requirements. Novelty: The proposed analytical method for the chiral analysis of linagliptin can be used by pharmaceutical industries quality control departments.

Separation and quantitative estimation of stereo-selective enantiomers of montelukast in pharmaceutical drug substance and tablets dosage forms by using stability-indicating normal phase-HPLC method.

Montelukast sodium (MLS) is a leukotriene receptor antagonist that relieves asthma, bronchospasm, allergic rhinitis, and urticaria. A simple, robust, and stability-indicating normal phase high-performance liquid chromatography method was developed to separate and quantitatively estimate the S-enantiomer of MLS. The chiral separation was achieved using USP L51 packing material along with a mobile phase consisting of a solvent mixture (n-hexane, ethanol, and propionic acid), a flow rate of 1.0mL/min, a detection wavelength of 284 nm, a column temperature of 30°C and an injection volume of 20 μL. The enantiomers peaks were well separated from the peaks of the placebo, diluting solvent, MLS, and its known impurities with a resolution of more than 2.2 and with no interference. Accuracy and linearity were studied in a range of 0.36-3.597 μg/mL (0.03%-0.30%), with good recoveries between 92.5% and 96.8% and a linear regression coefficient above 0.996. The suggested chiral chromatography method is being considered as an alternative and equivalent method to the United States Pharmacopeia and European Pharmacopeia monographs. The developed method was effectively employed for the study of release and stability samples of MLS. This HPLC method is also capable of separating and estimating the stereo-selective isomers (R- and S-enantiomers) of sulfoxide impurity of MLS in pharmaceutical medicine.

Why proanthocyanidins elute at increasing order of molecular masses when analysed by normal phase high performance liquid chromatography? Considerations of use

Although it is widely known that proanthocyanidins elute at an increasing order of molecular masses when analysed by normal phase high performance liquid chromatography (NP-HPLC), there is no a consistent explanation of the mechanisms of their separation until now. Therefore, the aim of the present study was to give a reliable response to this question, using a complex procyanidin-rich grape seed extract. For this, an off-column static simulation of extract injection and a fragmented-column dynamic procyanidin location tests were studied to show their precipitation in an aprotic solvent, besides another off-column static simulation and multiple contact dynamic solubilisation tests to confirm procyanidin redissolution in an aprotic/protic solvent system. The results showed that separation of procyanidins in the aprotic/protic solvent system of Diol-NP-HPLC was governed by precipitation/redissolution mechanism, that could be extended to all known plant proanthocyanidin homopolymers, including hydrolysable tannins, if they are able to accomplish this condition. However, separation of monomer species, namely catechins and some hydroxybenzoic acids, was based on classic adsorption/partition mechanism. Other factors, such as analyte solubility, chromatographic conditions and sample preparation, that affect the viability of proanthocyanidin analysis by NP-HPLC were stand out and guidelines for its durable and reproducible use were defined.

Preparation and chromatographic performance of chiral peptide-based stationary phases for enantiomeric separation.

This study presents the development of three new chiral stationary phases. They are based on silica modified with peptides containing phenylalanine and proline. Successful analyses and characterizations were conducted using Fourier transform infrared spectra, elemental analysis, and thermogravimetric analysis. After this, the enantioselective performance of the three chiral peptide-based columns was evaluated. The evaluation used 11 racemic compounds under normal-phase high performance liquid chromatography mode. Optimized enantiomeric separation conditions were established. Under these conditions, the enantiomers of flurbiprofen and naproxen were successfully separated on CSP-1 column: the separation factor of these was 1.27 and 1.21, respectively. In addition, the reproducibility of the CSP-1 column was also investigated. The results of the investigation illustrated that the stationary phases have good reproducibility (RSD = 0.73%, n = 5).

Chiral Analysis of the Key Intermediates of Tenofovir Alafenamide Fumarate

Abstract(R)-Tenofovir phenyl ester ((R)-1) and (R)-tenofovir diphenyl ester ((R)-2) are key intermediates for the practical synthesis of tenofovir alafenamide fumarate, which is a mainstay antiretroviral for the treatment of chronic hepatitis B and HIV-1 infections. This article deals with the chiral analysis of (R)-1 and (R)-2 against their respective optical impurity (S)-tenofovir phenyl ester ((S)-1) and (S)-tenofovir diphenyl ester ((S)-2) using a polysaccharide-coated chiral stationary phase (CSP) by normal-phase high-performance liquid chromatography (HPLC). To this end, a chiral synthetic strategy for (S)-2 was efficiently executed capitalizing on a classical Mitsunobu reaction to stereospecifically invert the configuration of chiral carbon in readily accessible (R)-HPA ((R)-4) to deliver (S)-HPA ((S)-4), from which (S)--tenofovir ((S)-3) was in turn prepared and further transformed into (S)-2. With reference substance (S)-2 in hand, a chiral analytical method for (R)-2 using Chiralpak AD-H as CSP by normal-phase HPLC has been developed and validated. The validation results indicated that this chiral analytical method has been achieved with satisfactory separation effect, high sensitivity, and good precision and accuracy, and thus can be deployed for the determination of optical impurities in samples of (R)-1 (via derivation to (R)-2) and (R)-2.

A direct and facile simultaneous quantification of non-polar and polar lipids in different species of marine samples using normal-phase HPLC–CAD

This study aimed to develop a sensitive and reliable normal-phase high-performance liquid chromatography–charged aerosol detector (NP-HPLC–CAD) method for direct and simultaneous quantification of diverse and complex lipid classes in marine samples. The 33-minute HPLC method so developed effectively separates 10 classes of non-polar and polar lipid. The quantitative calibration curves for all 10 lipid classes were built with satisfactory correlation coefficients (R2 ≥ 0.9910), wide linear range (0.01–5.00 mg/mL), low limit of detection (LOD, 2–40 ng) and limit of quantification (LOQ, 4–80 ng). The precision (relative standard deviation of 2.33–7.13 %) and recovery rate (83.69–108.06 %) for the quantitative method met the acceptance criteria of FDA guidelines. Finally, the NP-HPLC–CAD method was successfully used to analyze marine lipid samples from abalone, oyster, scallop, mussel and Antarctic krill. Results show complex lipid composition of marine samples, mainly containing triacylglycerol, phosphatidylethanolamine and phosphatidylcholine.

Glycosphingolipid Changes in Plasma in Parkinson's Disease Independent of Glucosylceramide Levels.

Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined. We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features. We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal-phase high-performance liquid chromatography assays; clinico-demographic data were extracted. Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b-series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a-series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels. Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF VENETOCLAX AND OBINUTUZUMAB BY USING NP-HPLC IN ACTIVE PHARMACEUTICAL INGREDIENT FORM

The aim of this study is to create and validate a fast, easy-to-use, affordable, sensitive, and accurate method for measuring Venetoclax and Obinutuzumab in bulk and pharmaceutical products using Normal-Phase High-Performance Liquid Chromatography (NP-HPLC). Hypersil chiral column was used with a running phase composed of Methanol: n-Hexane: 0.1% Formic acid (60:20:20 v/v) at a flow rate of 1.0 ml/min. UV detection was used at a wavelength of 225 nm. Obinutuzumab and Venetoclax correlation coefficients were found to be 0.999 over a concentration range of 6.25-37.5 µg/ml and 2.5-15 µg/ml, respectively. Venetoclax and Obinutuzumab have respective retention times of 3.134 and 6.929 minutes. The run time for separating Obinutuzumab and Venetoclax peaks was 10 minutes. This method, proposed as a regular analysis and quality control tool for medications that contain these active drugs either individually or in combinatio, was evident to be a suitable one. Keywords: HPLC, Obinutuzumab, Venetoclax, Development, Validation

Determination of malathion and its residues by normal-phase high-performance liquid chromatography method

Abstract The quality of pesticide formulations has an impact on the crop safety, environment and human health. Therefore, the development of new analytical methods for the determination of active substances in pesticide formulations in order to control their quality, as well as, their residues in food samples in order to ensure food safety, is always welcome. A new, simple, precise and accurate normal-phase high-performance liquid chromatography (NP-HPLC) method for determination of an active ingredient malathion in the commercial emulsifiable concentrate pesticide product has been developed and validated. The analysis was carried out on a LiChrosorb CN (250 x 4 mm, 5 μm) analytical column using isocratic elution with mobile phase consisted of n-hexane and dichloromethane (80/20, v/v), flow rate of 1 mL/min, constant column temperature at 25 °C and ultraviolet diode-array detection at 220 nm. The obtained values for multiple correlation coefficients (R 2 ≥ 0.9990), relative standard deviation of retention times, peak areas and heights (RSD ≤ 1.14%), recoveries ranged from 98.97 to 101.62%, revealed that the developed method has a satisfactory linearity, precision and accuracy. Also, the developed method was successfully applied for determination of malathion residues in apple juice samples, after preliminary sample preparation using solid-phase extraction. Specificity, selectivity, linearity, matrix effect, precision and accuracy were tested in order to validation of this method. The obtained results were in acceptable ranges and indicated that the developed method is suitable for routine determination of malathion in the pesticide formulation, as well as for determination of malathion residues in apple juice samples. The run time of HPLC analysis was about 6 min.

Stratum corneum ceramide profiles in vitro, ex vivo, and in vivo: characterization of the α-hydroxy double esterified ceramides.

The presence of a new ceramide subclass, the 1-O-acyl omega-linoleoyloxy ceramides [1-O-E (EO) Cer], has been previously highlighted in reconstructed human epidermis (RHE). These ceramides are double esterified on two positions. The first is the 1-O position of the sphingoid base moiety with a long to very long chain of acyl residues (1-O-E), and the second is the position of the ω-hydroxyl group of the fatty acid moiety with linoleic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. Thus, it is important to determine whether this subclass is also present in native human stratum corneum (SC). This work compares ceramide structures of this novel subclass between RHE (in vitro) and two sources of human SC (in vivo and ex vivo) using normal-phase high-performance liquid chromatography coupled to high-resolution mass spectrometry (NP-HPLC/HR-MSn). The results confirm the presence of this double esterified ceramide subclass [1-O-E (EO) Cer] in human SC. The molecular profile obtained from the RHE was very close to that found in the human SC (in vivo and ex vivo). In addition, thanks to the targeted MS2/MS3analysis, a new ceramide subclass was discovered and characterized in the three studied samples. We propose to name it [A-1-O-E (EO) Cer] because in these ceramides species, the fatty acid-esterified with the sphingoid base on the 1-O position-is hydroxylated on the α position. These results highlight the potential of both the analytical method and the characterization approach employed in this study.

Analytical Separation of Closantel Enantiomers by HPLC.

Closantel is an antiparasitic drug marketed in a racemic form with one chiral center. It is meaningful to develop a method for separating and analyzing the closantel enantiomers. In this work, two enantiomeric separation methods of closantel were explored by normal-phase high-performance liquid chromatography. The influences of the chiral stationary phase (CSP) structure, the mobile phase composition, the nature and proportion of different mobile phase modifiers (alcohols and acids), and the column temperature on the enantiomeric separation of closantel were investigated in detail. The two enantiomers were successfully separated on the novel CSP of isopropyl derivatives of cyclofructan 6 and n-hexane-isopropanol-trifluoroacetic acid (97:3:0.1, v/v/v) as a mobile phase with a resolution (Rs) of about 2.48. The enantiomers were also well separated on the CSP of tris-carbamates of amylose with a higher Rs (about 3.79) when a mixture of n-hexane-isopropanol-trifluoroacetic acid (55:45:0.1, v/v/v) was used as mobile phase. Thus, the proposed separation methods can facilitate molecular pharmacological and biological research on closantel and its enantiomers.

Simultaneous determination of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, sesamin, sesamolin, sesamol, and asarinin in sesame oil by normal-phase high performance liquid chromatography

A simple and fast normal-phase high performance liquid chromatography method for simultaneous detection of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, sesamin, sesamolin, sesamol, and asarinin in sesame oil (SO) was investigated. Conditions were a silica gel column as the separation unit, n-hexane and tetrahydrofuran (93:7, v/v) as the mobile phase, isocratic elution, a flow rate of 0.8 mL/min, column temperature of 30 °C, and florescence detector with excitation and emission wavelengths of 295 and 330 nm. SO samples were directly dissolved in n-hexane for HPLC analysis. Results showed that the method performed wide linear ranges and good linearity (0.27–100.0 μg/mL, R2 > 0.9996), low limits of detection (0.09–0.16 μg/mL) and quantification (0.27–0.41 μg/mL), good precision and accuracy (biases of <2.83 % and relative standard deviations of <3.77 %), good separation performance [resolution factor (1.66–6.20), separation factor (1.11–3.94), retention factor (1.08–1.42), and height of theoretical plates (0.01–0.04 mm)], and satisfactory recoveries (87.3–109.8 % for SO and 96.5–106.9 % for lard). The method was successfully applied for the simultaneous determination of the eight analytes in eleven SO samples.

Fast detection of adulteration of aromatic peanut oils based on alpha-tocopherol and gamma-tocopherol contents and ratio

A fast method based on normal-phase high-performance liquid chromatography with UV detection was applied to determine the tocopherols in aromatic peanut oil (APO) and four refined vegetable oils (soybean, sunflower, maize, and rapeseed oils). Our results showed that the tested oils varied greatly on the content of tocopherol isomers, especially on α- and γ-tocopherol concentrations. Consequently, (α+γ)-tocopherol (297.9–393.2 mg kg−1) and α-/γ-tocopherol (1.05–2.13) were successfully applied as discriminating parameters for detecting the adulteration of APO with above four refined vegetable oils. Furthermore, APO was mixed with the four oils (at the levels of 5, 10, 15, 20 and 25 g/100 g, w/w) to determine whether the tocopherol analysis is a better approach than fatty acid profiling for detecting APO adulteration. Our results showed that for tocopherols, our detection limits were 5 g/100 g for soybean oil and 10 g/100 g for other three oils in APO, showing a higher sensitivity than fatty acids profile based method for detecting APO adulteration. Thus, our newly developed method is an alternative and innovative tool for fast and cost-effective detection of the adulteration of APO with soybean, sunflower, maize, and rapeseed oils.

Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours

Normal-phase high-performance liquid chromatography (HPLC) is widely used in combination with evaporative light scattering detection (ELSD) for separating and detecting lipids in various food samples. ELSD responses of different lipids were evaluated to elucidate the possibilities and challenges associated with quantification by means of HPLC-ELSD. Not only the number and type of polar functional groups but also the chain length and degree of unsaturation of (free or esterified) fatty acids (FAs) had a significant effect on ELSD responses. Tripalmitin and trilinolein yielded notably different ELSD responses, even if their constituting free FAs produced identical responses. How FA structure impacts ELSD responses of free FAs is thus not predictive for those of triacylglycerols and presumably other lipids containing esterified FAs. Because ELSD responses of lipids depend on the identity of the (esterified) FA(s) which they contain, fully accurate lipid quantification with HPLC-ELSD is challenging and time-consuming. Nonetheless, HPLC-ELSD is a good and fast technique to semi-quantitatively compare the levels of different lipid classes between samples of comparable FA composition. In this way, lipid profiles of different flours from near-isogenic wheat lines could be compared.

Analysis of volatile oxidized oligomers from polyolefins by off-line normal phase high performance liquid chromatography and one-dimensional and comprehensive two-dimensional gas chromatography

The off-line coupled high performance liquid chromatography - gas chromatography - mass spectrometry (HPLC-GC–MS) was used for the analysis of oxidized oligomers from polyolefins. In order to optimize the HPLC fractionation method, a complex substance mixture of volatile organic compounds (VOC) from the provoked thermal oxidation of polyethylene (PE) was investigated. The fractionation was performed by normal phase HPLC, which separated the analytes according to their polarity. The seven obtained fractions were transferred off-line to the GC–MS. By means of fractionation nearly all coelutions present in the total extract could be avoided, which facilitated the identification of substances considerably. In addition, a separation according to functional moieties of substances was achieved, resulting in homologous series of compounds in the individual fractions: polyolefin oligomeric hydrocarbons (POH) in fraction 1, aldehydes and 7-, 6-, and 5-ketones in fraction 3, 2-ketones and alkenals in fraction 4, lactones and carboxylic acids in fraction 5, 1- and 2-alcohols in fraction 6 and diones in fraction 7, respectively. In this work, 7-, 6- and 5-ketones, δ-lactones, 2-alcohols and diones were identified for the first time in oxidized PE. Verification of fractionation and substance identification was done by comprehensive two-dimensional gas chromatography - time of flight - mass spectrometry (GCxGC-TOF-MS) measurements as well as off-line HPLC-GC–MS measurements using a mixture of reference substances. The fractionation approach was applied for the analysis of a commercial PE film after melt flow extrusion. POH were eluted in fraction 1, while n-aldehydes (with a carbon chain length of C11-18) were identified in fraction 3 and 2-ketones (C13-15) in fraction 4. The amounts of the individual detectable oxidized oligomers were estimated semi-quantitatively to range from 10 – 170 µg/kg.

Therapeutic drug monitoring of olanzapine: Easy and reliable method for clinical correlation

AIM:The current work establishes an easy, reliable technique for the estimation of serum Olanzapine concentration which correlates it clinically.SUBJECTS AND METHODS:The work was agreed in 61 schizophrenic patients who were on olanzapine. Serum drug amount was estimated by normal-phase high-performance liquid chromatography and brief psychiatry rating scale was used to determine disease progression.RESULTS:Samples provided 61 patients, 40 were under sub-therapeutic range, 18 were under therapeutic range and 3 were above the therapeutic range.CONCLUSION:Therapeutic drug monitoring must be a part of clinical practice in psychiatric hospitals for optimizing the dose of an individual patient along with the correlation of serum concentration with the clinical assessment scales.

Determination of biodiesel and used cooking oil in automotive diesel/green diesel fuels through high-performance liquid chromatography.

This study reports a simple and convenient analytical method for the simultaneous determination of biodiesel and vegetable oils or used cooking oils in petrodiesel and green diesel (hydrotreated vegetable oils or paraffinic diesel). The approach is based on normal-phase high-performance liquid chromatography with refractive index detection. It employed silica stationary phase, n-hexane mobile phase with isopropanol modifier to achieve optimum separation between hydrocarbons (petrodiesel or green diesel), fatty acid methyl esters (biodiesel) and triglycerides (vegetable oils and used cooking oil). In addition to determining vegetable oils or used cooking oils as adulterants in diesel, this method is also proposed as a better alternative to the standard method ASTM D7371, which is currently recommended for determining fatty acid methyl esters in petrodiesel. The method development involved screening of various stationary and mobile phases, with and without modifiers, to achieve acceptable chromatographic resolutions between analytes. Under the optimized method conditions, silica column, and n-hexane containing 0.6% isopropanol as the mobile phase provided the best results. The real-world scenario was simulated for the method validation carried out by fortifying Jatropha seed oil, soybean oil, and used cooking oil in the biodiesel blended petrodiesel and green diesel. Measurement of all analytes was accompanied by high precision, low limit of detection/quantification and linear response range of 0.05 to 50% for biodiesel, and 0.05 to 30% for vegetable oils.The proposed method is simple, fast (runtime 7min), and does not require sample pre-treatment and backflushing.