Gradient Reversed-phase High-performance Liquid Chromatography Research Articles

A stability-indicating method development and validation for the determination of related substances in novel synthetic decapeptide by HPLC.

In the present scenario, peptide is an emerging field of research having vast therapeutic applications. Diverse impurities may rise from various stages of the synthesis process and storage of the peptides. Because these contaminants may have an impact on the therapeutic safety and effectiveness of peptides in their approaching applications, they must be identified and carefully monitored. Considering the pharmaceutical importance of the extent of peptides, we were motivated to synthesize a decapeptide and establish a novel gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method for its analysis along with efficient separation of its six related impurities. Different buffers, organic modifiers, and columns were used in the tests for good separation of these impurities. To establish a stability-indicating method, a stress study was also conducted. The International Conference on Harmonization (ICH) guidelines have been followed for validation of the developed analytical method. The validated method revealed sufficient accuracy, specificity, linearity, robustness, precision, and high sensitivity for its intended use. The proposed method could be appropriate for routine analysis and stability assessment of the decapeptide, which might be useful for further scientific investigation.

Validation of HPLC-DAD Based Stability Indicating Assay Method for Ornidazole in Periodontal Polymeric Hydrogel: Robustness Study using Quality by Design (QbD) Approach

In the present study, a gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method has been designed and validated to quantify Ornidazole (OZ) in the marketed formulation (oral gel) with the application of QbD. The proposed HPLC method utilizes an RP C18 column (4.6 x 250 mm, 5 μm). The mobile phase contained HPLC grade Solvent A (water) and Solvent B (acetonitrile) and ran at a flow rate of 1 mL/min in a gradient mode. The linearity was plotted in an OZ range of 1–12 μg/mL and the correlation coefficient was found to be 0.9998. The limit of quantification, as well as the limit of detection, has been recorded as 0.70 μg/mL and 0.23 μg/mL. Inter- and intra-day precision (RSD) percentages were reported respectively within a range of 0.262–0.589 and 0.179–0.879. At 80, 100, and 120 percent different OZ levels, the average accuracy was 99.55, 99.58, and 99.92, respectively. The result of the study indicates that the OZ gel estimate HPLC method has good sensitivity, reproducibility, accuracy, and precision. This method may be used for the estimation of OZ in a gel formulation in quality control laboratories.

A Novel Approach for Assessing Technical Grade and Quality of Lambda-Cyhalothrin and Acetamiprid in Insecticides Used in Agricultural Systems by HPLC Technique in Southern Benin

In Benin, synthetic insecticides are the main pest control option used by farmers to protect and enhance their production. However, failures to control the target pests are often observed after application and may be related to agricultural practices or insecticide quality. The present work was designed to assess a rapid, simple, and reliable analytical method for detecting and quantifying the most commonly used insecticides (λ-cyhalothrin and acetamiprid) in Benin. The analytical standard technical grade separation was performed by gradient reversed-phase high-performance liquid chromatography on a C18 stationary-phase column. The mobile phase consisted of a mixture of acetonitrile/water using a gradient flow. The flow rates were 1 and 1.4 mL·min−1 for λ-cyhalothrin and acetamiprid, respectively. The analysis times were 15 and 20 min, with retention times of 2.35 and 7.94 min for λ-cyhalothrin and acetamiprid, respectively. Results reveal that most of the surveyed farmers were not educated (70% < Primary School Certificate) and were men (95%). Of the main insecticides applied by farmers, λ-cyhalothrin and acetamiprid were found to be the most technical-grade ones. Furthermore, the analysis of insecticides showed that the concentrations obtained in our study often differed from the ones mentioned on insecticide labels. The proposed method is useful for quantifying insecticides in various technical and commercial formulations with little interference from additives.

Normal-Phase TLC and Gradient Reversed-Phase HPLC for the Simultaneous Determination of Enrofloxacin and Bromhexine HCl in Presence of Two of Their Official Impurities.

In this work, two chromatographic methods are developed and validated for the determination of enrofloxacin and bromhexine (BRM) HCl in the presence of two of their specified impurities, ciprofloxacin and BRM impurity C. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high-performance liquid chromatography (HPLC). In case of TLC-densitometry, good separation was achieved by using mobile phase of n.butanol:acetone:water:glacial acetic acid:triethylamine (10:3:1:0.5:0.5, by volume) on silica gel stationary phase at 254-nm detection. The developed HPLC method used BDS HYPERSIL C18 column with a mobile phase of water:acetonitrile:methanol:triflouroacetic acid. A linear gradient elution of 75-10%, 20-50% and 5-40% for water, acetonitrile and methanol, respectively, was applied in 13min at a flow rate of 1.5mLmin-1. These methods were sufficient to separate the four substances simultaneously, and they are validated as per International Conference on Harmonization guidelines.

Normal-Phase TLC and Gradient Reversed-Phase HPLC for the Simultaneous Determination of Enrofloxacin and Bromhexine HCl in Presence of Two of Their Official Impurities

In this work, two chromatographic methods are developed and validated for determination of enrofloxacin (ENR) and bromhexine HCl (BRM) in the presence of two of their specified impurities, ciprofloxacin (CIP) and BRM impurity C. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high performance liquid chromatography (HPLC). In case of TLC- densitometry, good separation was achieved by using mobile phase of n.butanol: acetone: water: glacial acetic acid: triethylamine (10:3:1:0.5:0.5, by volume) on silica gel stationary phase at 254nm detection. The developed HPLC method used BDS HYPERSIL C18 column with a linear gradient elution of 75-10%, 20-50% and 5-40% for water, acetonitrile and methanol, respectively, in 10 min at a flow rate of 1.5 mL min-1.These methods were sufficient to separate the four substances simultaneously and they are validated as per International Conference on Harmonization (ICH) guidelines.

Validated stability-indicating RP-HPLC method for simultaneous estimation of cilnidipine and chlorthalidone in tablet dosage form

A novel, simple, specific, accurate & precise stability-indicating Gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous estimation of Cilnidipine & Chlorthalidone in tablet dosage form, validated as per ICH guideline. The separation was achieved on Inertsil ODS column (250 mm x 4.6 mm, 5 μm) in a gradient mode. The mobile phase consisted of Methanol, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10% v/v Ortho Phosphoric Acid (50:50 v/v) (Solution A) and Acetonitrile, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10%v/v Ortho Phosphoric Acid (75:25 v/v) (Solution B), gradient programming for 20 min at 1 ml/min rate of flow and response was detected at 225 nm. The retention time was found to be 3.580 min and 12.606 mins for Chlorthalidone and Cilnidipine, respectively. The method is validated according to ICH guideline, which includes linearity, specificity, accuracy, precision and robustness. Linearity was obtained over the concentration range of 10-60 μg/ml for Cilnidipine and 6.25-37.5 μg/ml for Chlorthalidone, had a regression coefficient (r2) almost 0.9966. The % Recovery was found to be 99.63-100.59 % and 100.24-100.51 % for Cilnidipine and Chlorthalidone, respectively. The method was found to be specific enough to separate all degradation products from the active compound. Drug samples were exposed to various stress conditions like photolysis, oxidation, heat conditions, and hydrolysis (acidic and alkaline), there was no interference of any degradants and excipient in the determination of drugs so that methods can be successfully applied for routine QC analysis.

Vliyanie zamorazhivaniya na dinamiku aminokislotnogo sostava belkov telyatiny

The results of study of effect of freezing temperature on the change of amino acid content and the ratio of essential and nonessential amino acids in veal proteins are given. The meat of calves at the age of 3 months, bred in the Leningrad region of the RF, was the object of research. The amino acid composition of veal proteins was analyzed using the Shimadzu 20AD Chromatograph with precolumn phenylisothiocyanate derivatization and spectrophotometric detection at 254 nm by reversedphase gradient highperformance liquid chromatography (HPLC). The Kjeldahl method was used to define the protein content on the basis of the sample mineralization. It was shown that the change of the amount and ratio of essential and nonessential amino acids of veal proteins depended on the freezing temperature as well as on structural features of Rside radicals. Also it was established that the total amount of essential and nonessential amino acids in veal proteins during freezing at -24 and -35 °С temperatures decreased by 13,5 and 8,2%, respectively; the total of essential amino acids by 11 an 7%, nonessential ones by 22 and 11%, respectively. It was proved that significant changes were characteristic of amino acids with polar anionic and cationic radicals. So, the amount of amino acids with polar negatively charged radicals during veal freezing at temperatures -24 and -35 ° С decreased by 22 and 16,5%; with polar positively charged radicals - by 17 and 13,5%, respectively. At the same time, the freezing temperature affects slightly the change in the content of amino acids with nonpolar (hydrophobic) Rgroups and polar nonionogenic radicals. It was shown that the total amount of free amino acids increased independently of the freezing temperature. However, more essential amino acids were accumulated in veal frozen at a temperature of -24 ° С (2,33 times) than at -35 ° С (1,80 times). The amount of free nonessential amino acids changed to a lesser extent

SIMULTANEOUS EVALUATION OF ABACAVIR SULFATE AS WELL AS LAMIVUDINE IN MEDICAL FORMULATIONS BY GRADIENT REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY TECHNIQUE

Objective: A precise, accurate, simple, and gradient reversed-phase high-performance liquid chromatography (HPLC) method was adapted for the determination of abacavir sulfate (ABV) in combination with lamivudine (LMV) having tablet formulations simultaneously. This method developed has been validated as per the guidelines of ICH.Method: Waters HPLC has been used in the method with a column named Zorbax C18 with the dimensions as 4.6 nm×150 mm, 3.5 μm. Phosphate buffer (PH - 3.9) was used as Eluent - A, Eluent - B was methanol, and water and methanol (50:50 v/v) were utilized as diluents. The rate of flow was 1.5 ml/min.Results: The wavelength of detection has been detected at about 270 nm. Linearity ranges of ABV and LMV were 88–266 μg/ml and 38–116 μg/ml, respectively. Retention times of ABV (3.66 min) and LMV (10.71 min) were determined. The values of the study of percentage recovery of ABV and LMV were determined to be within 98.3–99.2%.Conclusion: The estimation of ABV and LMV in all pharmaceutical dosage forms could be performed successfully by employing this method.

HPLC Method for Simultaneous Determination of Bisphenol-A-Diglycidyl Ether and Some of Its Reaction Products in Canned Foods Using Photodiode Array Detector.

Gradient reversed-phase high-performance liquid chromatography with photodiode array detection was used for separation, detection and quantification of bisphenol-A-diglycidyl ether (BADGE) and some of its reaction products, namely, BADGE·HCl·H2O, BADGE·H2O and BADGE·2HCl in pure form and in canned foods, where canned beans and tuna were used as representatives of aqueous and oil-in-water food matrices, respectively. The proposed method had a linear range of 0.01-0.5 μg g-1 for BADGE·HCl·H2O, BADGE·H2O, BADGE·2HCl and 0.02-0.7 μg g-1 for BADGE in aqueous food matrices. In oil-in-water matrices, the method was proven to be sensitive over a linear range of 0.01-0.5 μg g-1 for BADGE·HCl·H2O, BADGE·H2O and 0.02-0.7 μg g-1 for BADGE·2HCl and BADGE. The limits of detection and quantification ranged from 0.24 to 1.22 ng g-1 and 0.73 to 14.07 ng g-1, respectively. Excellent intraday and interday precision (n = 9) were obtained with RSD% of 0.84-2.19% and 1.88-2.52%, respectively. Accuracy was measured at five concentration levels and the recoveries ranged from 96.31% to 98.76% with an acceptable variation of ±0.9-2.87. Results suggest that the proposed method could be applied for the routine analysis of the studied compounds in their laboratory-prepared mixtures and in various types of canned foods following the limits and regulations of the European Union.

Abstract 738: Development of an HPLC method for the assessment of hydrophobicity of ADC payloads

Abstract Antibody-Drug Conjugates (ADCs) are antibodies attached to cytotoxic agents (payloads) through chemically labile linkers. The development of ADCs can fail at various stages, a common cause being a lack of reproducibility in the methods used to attach the payload to the antibody (i.e., the conjugation process) to provide a good yield and uniform drug-antibody ratio (i.e., the DAR). One of the major problems associated with conjugation is the hydrophobicity of a payload which can cause aggregation. The aim of this study was to create a hydrophobicity model to study the physicochemical properties of payloads at an early stage. This should allow their hydrophobicity to be evaluated and optimised at the design stage. A predictive model has been developed based on High Performance Liquid Chromatography (HPLC). Initial work involved development of a gradient reversed-phase HPLC method, and its use in the analysis of multiple reference compounds to establish their relative retention times. This involved the evaluation of a range of reversed-phase columns and solvent systems in order to obtain reproducibility. These initial studies, which utilized a C18 monolithic column for optimum results, demonstrated a clear relationship between the retention time (RT) of the reference compounds and their experimental LogP values obtained from the literature (r = 0.983). Next, a number of commercially available ADC payloads were analysed using the same HPLC method to determine their RT values, and their LogP values were calculated using the ChemDraw software. Payloads studied included Monomethyl Auristatin E (MMAE), Talirine and Tesirine (PBD dimers), Mertansine (DM1) and Calicheamicin. For this group of compounds, the results confirmed that their RT values correlated well with their calculated LogP values (r = 0.94). For example, the payload Talirine, which has a calculated LogP of 3.77, had an RT value of 17 min on the HPLC system equating to a LogP of 3.13 based on the reference compounds. Finally, some novel DNA-interactive payloads (e.g., the pyridinobenzodiazepine FGX-2-62) were evaluated using the same methodology. For FGX-2-62, the calculated LogP varied significantly between different software packages (i.e., 1.47, 3.38 or 4.46 for ChemSketch, MarvinSketch or ChemDraw, respectively). However, its HPLC retention time using the same method was 1.88 min, equating to a LogP of 1.63. This suggests that FGX-2-62 is significantly less hydrophobic than Talirine (e.g., RTs of 1.88 min versus 17 min, respectively), an observation consistent with the ease of conjugation of FGX-2-62 to antibodies without significant aggregation. Overall, these results suggest that HPLC retention time derived from a suitable HPLC method is more reliable than the currently available software-based LogP prediction methods in providing a realistic assessment of the hydrophobicity of novel payloads and their propensity to cause aggregation during conjugation. Citation Format: Ilona Pysz, Paul J. Jackson, Khondaker M. Rahman, David E. Thurston. Development of an HPLC method for the assessment of hydrophobicity of ADC payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 738.

Separation of hydrophobically modified hyaluronic acid according to the degree of substitution by gradient elution high performance liquid chromatography.

Amphiphilic hyaluronic acid (HA), synthesised by modifying HA to varying extents with acrylate groups, was successfully separated according to degree of substitution (DS) using solvent gradient high performance liquid chromatography (HPLC). Two HPLC methods based on the amphiphilic structure of the HA were developed. In the first approach, normal phase gradient HPLC was explored, and separation was based on the interactions of HA's polar hydroxyl groups with a polar cyano stationary phase. In the second approach, separation was based on the interaction of the hydrophobic acrylate moieties with a non-polar C8 stationary phase (reversed phase gradient HPLC). The separation was optimised by using an electrolyte in the sample solvent to suppress non-covalent interactions and improve the selectivity of the developed method. The photolytic stability of the modified and unmodified HA was also investigated in order to optimise the sample preparation procedure. Furthermore, an alternative method to NMR spectroscopy was developed for determining the DS of HA. Graphical abstract ᅟ.

Characterization of stress degradation products of amodiaquine dihydrochloride by liquid chromatography with high-resolution mass spectrometry and prediction of their properties by using ADMET Predictor™.

The degradation behavior of amodiaquine dihydrochloride, an antimalarial drug, was investigated in solution as well as solid states. The drug was subjected to hydrolytic, photolytic, oxidative, and thermal stress conditions, according to International Conference on Harmonization guideline Q1A(R2). It showed extensive hydrolysis in acidic, alkaline, and neutral solutions both with and without light, while it proved to be stable to thermal and oxidative conditions. In total, six degradation products were formed, which were separated on a C8 column, employing a gradient reversed-phase high-performance liquid chromatography method in which acetonitrile and 10mM ammonium formate (pH 3.0) were used in the mobile phase. To characterize the degradation products, mass fragmentation behavior of the drug was established by direct infusion of solution to quadrupole time-of-flight and multiple-stage mass spectrometry systems. Liquid chromatography with high-resolution mass spectrometry studies were subsequently carried out on the stressed samples using the same gradient high-performance liquid chromatography method employed for the separation of the degradation products. Hydrogen/deuterium exchange studies were additionally conducted to determine the number of labile hydrogen atoms. The degradation pathway of the drug was delineated, justified by mechanistic explanation. Lastly, ADMET Predictor™ software was employed to predict relevant physicochemical and toxicity data for the degradation products.

Development and Optimization of HPLC Analysis of Metronidazole, Diloxanide, Spiramycin and Cliquinol in Pharmaceutical Dosage Forms Using Experimental Design.

A new simple, sensitive, rapid and accurate gradient reversed-phase high-performance liquid chromatography with photodiode array detector (RP-HPLC-DAD) was developed and validated for simultaneous analysis of Metronidazole (MNZ), Spiramycin (SPY), Diloxanidefuroate (DIX) and Cliquinol (CLQ) using statistical experimental design. Initially, a resolution V fractional factorial design was used in order to screen five independent factors: the column temperature (°C), pH, phosphate buffer concentration (mM), flow rate (ml/min) and the initial fraction of mobile phase B (%). pH, flow rate and initial fraction of mobile phase B were identified as significant, using analysis of variance. The optimum conditions of separation determined with the aid of central composite design were: (1) initial mobile phase concentration: phosphate buffer/methanol (50/50, v/v), (2) phosphate buffer concentration (50 mM), (3) pH (4.72), (4) column temperature 30°C and (5) mobile phase flow rate (0.8 ml min-1). Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9999. Limits of detection for all of the analyzed compounds ranged between 0.02 and 0.11 μg ml-1; limits of quantitation ranged between 0.06 and 0.33 μg ml-1 The proposed method showed good prediction ability. The optimized method was validated according to ICH guidelines. Three commercially available tablets were analyzed showing good % recovery and %RSD.

Assessment and Separation of Angiotensin I-Converting Enzyme Inhibitory Peptides in Chinese Soypaste

Abstract A Chinese soypaste-derived fraction with potent angiotensin I-converting enzyme (ACE) inhibitory activity (IC50 = 25.9 μg/mL) was obtained by treating soypaste extract with 80% ethanol. The result of gradient reversed-phase high-performance liquid chromatography (RP-HPLC) suggested that bioactive peptides bearing some polarity groups made a substantial contribution to the ACE inhibitory activity. By mass spectrometric analysis, a component was separated as Glu-Ser-Gly-Asp which was then found to act in a dose-dependent manner against ACE activity as a non-competitive inhibitor, with an IC50 value of 2.297 mM.

Three-dimensional desirability spaces for quality-by-design-based HPLC development.

In this study, three-dimensional desirability spaces were introduced as a graphical representation method of design space. This was illustrated in the context of application of quality-by-design concepts on development of a stability indicating gradient reversed-phase high-performance liquid chromatography method for the determination of vinpocetine and α-tocopheryl acetate in a capsule dosage form. A mechanistic retention model to optimize gradient time, initial organic solvent concentration and ternary solvent ratio was constructed for each compound from six experimental runs. Then, desirability function of each optimized criterion and subsequently the global desirability function were calculated throughout the knowledge space. The three-dimensional desirability spaces were plotted as zones exceeding a threshold value of desirability index in space defined by the three optimized method parameters. Probabilistic mapping of desirability index aided selection of design space within the potential desirability subspaces. Three-dimensional desirability spaces offered better visualization and potential design spaces for the method as a function of three method parameters with ability to assign priorities to this critical quality as compared with the corresponding resolution spaces.

DETERMINATION OF BUTORPHANOL USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY IN SMALL VOLUME PLASMA SAMPLES

A high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of butorphanol in bearded dragon plasma. Following a liquid extraction using ethyl acetate and hexane, samples were separated by gradient reverse phase HPLC on a Symmetry C18 column and quantified using fluorescence detection at an excitation of 200 nm and an emission of 325 nm. The mobile phase was a mixture of 0.05 M ammonium acetate (pH, 4.1) and acetonitrile, with a flow rate of 1.3 mL/min. A linear curve was produced over the concentration range of 2.5 to 1500 ng/mL. Intra- and inter-assay variability for butorphanol were less than 10% and the average recovery was 90%. The new assay quantifies therapeutic levels of butorphanol in 100 µL samples from pharmacokinetic studies conducted at this institution.

Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core–shell stationary phase

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core–shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

Determination of lipophilicity for antitumor acridinone derivatives supported by gradient high-performance liquid chromatography method

Abstract The lipophilicity values of selected acridinone (imidazoacridinone and triazoloacridinone) derivatives were measured by gradient reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 stationary phase with a water/acetonitrile mixture as a mobile phase. The retention times obtained served as input data and appropriate log kw values (i.e., the retention factor log kw extrapolated to 0% organic modifier) as an alternative to log P were calculated using the DryLab program. The relationships between the lipophilicity (log kw) and the chemical structure of the studied compounds, as well as correlation between experimentally determined lipophilicities (log kw) and log P data calculated using some commonly available software, are discussed.

On the utility of predictive chromatography to complement mass spectrometry based intact protein identification

The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

Improved Analytical Extraction and Clean-up Techniques for the Determination of PAHs in Contaminated Soil Samples

Development and optimisation of fast, efficient, quantitative, economic and environmentally friendly analytical extraction techniques for the extraction clean up, and pre-concentration in the quantification of 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs) in contaminated soils have been carried out. Three different extraction methods (Soxhlet, ultra sonication and mechanical shaking) were investigated on a low-level PAH soil certified reference material (CRM131-100) and the results were compared to determine the technique with the highest extraction efficiency. The clean up and pre-concentration procedures were optimized using both the conventional method (i.e. column packing with silica gel) as well as the solid phase extraction (SPE). Chromatographic conditions for the separation of PAHs using High Performance Liquid Chromatography (HPLC) using UV-DAD and fluorimetric detection with programmed excitation and emission wavelengths were also optimized. Six different extraction solvents: acetone, cyclohexane, 2- propanol, methanol, acetonitrile and dichloromethane, were tested to select the most suitable solvent for the extraction of the 16 PAHs from the certified soil reference material. Acetonitrile, dichloromethane and tetrahydrofuran were also tested as eluants for the optimisation of SPE clean up. The optimized ultrasonic extraction procedure utilizing four 15 minutes extraction cycles at 50 oC and SPE clean up with tetrahydrofuran: acetonitrile (1:1) and subsequent separation by gradient reversed phase HPLC with fluorimetric detection extracted the PAHs from the certified reference material with recoveries ranging from 63.6 % to over 100 % .