Phase Ultra Performance Liquid Chromatography Research Articles

Determination of nine estrogenic steroids in milk using matrix solid phase dispersion-ultra performance liquid chromatography with mass spectrometric detector

An analytical method for the multiresidue determination of nine estrogenic steroids in milk was developed by modified matrix solid phase dispersion (MSPD) purification and ultra performance liquid chromatography (UPLC) with mass spectrometric detector (MSD). The sensitivity and accuracy of MSD were better than that of ultraviolet detector. In comparison with traditional mass spectrometry, the merits of MSD were simpler in operation and shorter in starting time (5 min). The results showed that the limits of detection of the compounds with nucleophilic substitution were high in positive ion mode of MSD and were easily affected by environmental conditions. The matrix effects of milk samples reduced from 84%-160% to 80%-121% after MSPD purification. The intraday precision and interday precision of the nine estrogenic steroids were 0.87%-1.78% and 1.82%-3.79%, respectively. The average recoveries were 68.7%-94.7%, and the relative standard deviations (RSDs) were less than 10%. The limits of detection (LODs) were 0.5-10 μg/kg. The limits of quantification (LOQ) were 2-20 μg/kg.

Low temperature freezing followed by dispersive solid phase extraction for the determination of 104 pesticide residues in vegetable oils using ultra-performance liquid chromatography-tandem mass spectrometry

A method using liquid-liquid extraction (LLE) followed by clean-up steps of centrifugation, freezing and dispersive solid phase extraction (D-SPE) and ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS) has been devel- oped for the determination of trace levels of 104 pesticides in vegetable oils. LLE has been optimized to extract these pesticide residues from oils to obtain the highest recoveries of pesticides and the lowest co-extract fat residue in the final extract. In addition, the centrifugation and freezing steps as well as D-SPE with primary secondary amine (PSA), graphite carbon black (GCB) and C18 were used as the clean-up steps to minimize the co-extract fat. The recoveries obtained ranged from 55% to 121% and the relative standard deviations (RSDs) ranged from 0. 47% to 19. 2% at the spiked levels of 0.01, 0.02 and 0.05 mg/kg. This method, combining with accurate and sensitive detection, allowed quantification and confirmation at levels as low as 1 µg/kg for 80% of the analytes. The limits of quantification (LOQs) of the most compounds were below the maximum residue limits (MRLs) established by the Chinese legislations for oils. The proposed method was applied successfully for the residue determination of the selected pesticides in oils.

Evaluating Nothapodytes nimmoniana population from three localities of Western Ghats using camptothecin as phytochemical marker and selection of elites using a new-content range chart method.

Background:Nothapodytes nimmoniana (Grah.) Mabb. is a high valued medicinal plant endemic to Western Ghats of India, distributed in fragmented populations. The plant is valued for potent anticancer drug camptothecin (CPT).Objective:The study compares and expounds variation in CPT content from leaves and stems of N. nimmoniana obtained from three populations of Western Ghats, India. The study also describes a method for categorizing these populations using content range chart (CRC) method for percent yield of CPT.Materials and Methods:A total of 60 samples were investigated including ten each of leaves and stems from three localities. Micro-extraction method was implemented to extract CPT. reversed phase ultra-performance liquid chromatography photo diode array technique was used to quantify CPT.Results:Leaf samples of an individual collected from Joida, yielded lowest CPT content (0.002 ± 0.000 g/100 g), whereas a stem sample from Amgaon, yielded highest CPT content (0.123 ± 0.006 g/100 g). The findings suggest great variation in individuals producing and accumulating CPT. Using this data along with earlier published work, five categories of CPT yielding plants were made viz. I: Very low: <0.020, II: Low: 0.021-0.039, III: Moderate: 0.040-0.059, IV: High: 0.060-0.079 and V: Very high: >0.080. Based on CPT content in leaves, majority of individuals were under very low category (Ist) and on the other hand stem samples were in ‘II’ category. Besides, very few individuals were observed in category ‘V’.Conclusion:The study expounds use of CRC method for identifying elite population and suggests the need for its conservation.

DEVELOPMENT AND VALIDATION OF RP-UPLC METHOD USING EXPERIMENTAL DESIGN APPROACH FOR SIMULTANEOUS ESTIMATION OF CANDESARTAN CILEXETIL AND CHLORTHALIDONE

Candesartan Cilexetil is an antihypertensive agent currently available in combination with Hydrochlorothiazide. However, it has been proven by research that half dose of Chlorthalidone is equipotent to the dose of Hydrochlorothiazide in similar combination with Candesartan Cilexetil. At present there is no Liquid Chromatographic (LC) method available for the simultaneous estimation of Candesartan Cilexetil and Chlorthalidone in pharmaceutical dosage form. A simple, rapid, reliable and robust reversed phase ultra-performance liquid Chromatography (RP-UPLC) method was developed as per International Conference on Harmonization (ICH) guidelines. The best separation was achieved in less than 5 minutes on a 50 × 2.1 mm, 2.2 µm particle size Dionex C18 column with the gradient mobile phase 5 mM, 6.2 ± 0.5 pH ammonium acetate buffer - acetonitrile at a flow rate of 0.5 mL/min at 215nm. The detector response was linear in the range of 10-200 ppm of these drugs. LOD obtained was 3.04 ppm for Candesartan Cilexetil, 2.82 ppm for Chlorthalidone. Further evaluation of robustness of this method was carried out using Plackett-Burman verification with four factors two levels (high &amp; low) within analytical QbD-principles using statistical software. The outcome of normal probability and overall desirability plots were found acceptable.

Method Development and Validation for the Determination of Potential Impurities Present in Azilsartan medoxomil Tablets by Reverse Phase-Ultra Performance Liquid Chromatography

The present paper describes about precise, sensitive, rapid method for the determination of five potential impurities present in Azilsartan medoxomil Tablets. Separation of impurities at satisfactory level is achieved in Acquity UPLC BEH C18, 100 mm length x 2.1 mm id with 1.7 µm particle size column. Mobile phase A consists of 0.1% ortho phosphoric acid in water adjusted the pH to 3.0 with dilute sodium hydroxide and acetonitrile as Mobile phase B using gradient elution mode. Flow rate was kept at 0.5 mL.min-1 with a monitoring wavelength of 215 nm. Developed method was successfully validated as per method validation parameters recommended by International Conference on Harmonisation (ICH) for specificity, linearity, precision, accuracy, determination of LOD, LOQ, solution stability and robustness. The validated Reverse phase-Ultra Performance Liquid Chromatography (RP-UPLC) method was successfully used for quantitative determination of potential known and unknown impurities of Azilsartan medoxomil in Azilsartan medoxomil tablet dosage form, helping to identify the therapeutic efficacy at shorter run time.

Development and validation of a UPLC method for screening potentially counterfeit anti-hypertensive drugs using design of experiment

An isocratic reversed phase ultra performance liquid chromatography (RP-UPLC) method was developed for screening counterfeit medicines with UV detection at 210 nm.

Simultaneous determination of sixteen perfluorinated organic compounds in surface water by solid phase extraction and ultra performance liquid chromatography with electrospray ionization tandem mass spectrometry

A high-throughput detection method has been developed for the determination of sixteen perfluorinated organic compounds (PFCs) in surface water by solid phase extraction-ultra performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (SPE-UPLC-ESI-MS/MS). The water samples were concentrated and purified through WAX solid phase extraction cartridges. The UPLC separation was performed on an ACQUITY UPLC BEH C18 column utilizing a gradient elution program of methanol (containing 2 mmol/L ammonium acetate) and water (containing 2 mmol/L ammonium acetate) as the mobile phases at a flow rate of 0.4 mL/min. The MS/MS detection was performed under negative electrospray ionization ( ESI ) in multiple reaction monitoring (MRM) mode. Good linearities were observed in the range of 0.5-100 gg/L or 1.0 - 100 microg/L with correlation coefficients from 0.998 7 to 0.999 9. The limits of detection (LODs) for the sixteen perfluorinated organic compounds were in the range of 0.06-0.46 ng/L. The recoveries ranged from 67.6% to 103% with the relative standard deviations between 2.94% and 12.0%. This method was characterized by high sensitivity and precision, extensive range and high speed, and can be applied for the analysis of PFC contaminants in surface water.

A validated stability-indicating UPLC method for determination of diclofenac sodium in its pure form and matrix formulations

The aim of this work is to develop a validated stability indicating reverse phase ultra performance liquid chromatography (UPLC) method for the rapid and accurate determination of diclofenac sodium in its pure form and in matrix formulations. This UPLC method is composed of isocratic mobile phase, 0.05M ammonium acetate buffer (pH=2.5) and acetonitrile (50:50), with flow rate 0.5ml/min, column BEH C18 (2.1×50mm, 1.7μm). The method is rapid (1.2min run), selective with well resoluted diclofenac peak with retention time 0.94min and sensitive (LOD=2ppm and LLOQ=6ppm) with UV detection at 254nm. The drug was subjected to acidic, alkaline media, boiling and oxidizing agent to apply stress conditions. The developed method was able to separate degradation product generated under forced degradation studies. The developed method was validated as per the FDA guidelines for specificity, linearity, accuracy, precision, LOD, LLOQ and found to be satisfactory. The study suggests that the developed UPLC method can be used for the assessment of drug purity and stability. It can be also used to monitor the drug content and release from different formulations without any interference of excipients and/or degradation products.

Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography–mass spectrometry

A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.

Use of UPLC-ESI-MS/MS to quantitate free amino acid concentrations in micro-samples of mammalian milk

Although free amino acids (FAA) account for a small fraction of total nitrogen in mammalian milk, they are more abundant in human milk than in most formulas, and may serve as a readily available source of amino acids for protein synthesis, as well as fulfill specific physiologic roles. We used reversed phase Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) technique for FAA profiling in milks from three species (human, rat and cow) with a simple and rapid sample preparation. The derivatization procedure chosen, combined with UPLC-ESI-MS/MS allowed the quantitation of 21 FAA using labeled amino acids (Internal Standards) over a 10 min run time in micro-samples of mammalian milk (50 μL). The low limit of quantitation was 0.05 pmol/μL for most FAA with good repeatability and reproducibility (mean CV of 5.1%). Higher levels of total FAA were found in human (3032 μM) and rat milk (3460 μM) than in bovine milk (240 μM), with wide differences in the abundances of specific FAA between species. This robust analytical method could be applied to monitor FAA profile in human breast milk, and open the way to individualized adjustment of FAA content for the nutritional management of infants.

Development and validation of stability-indicating UPLC method for the determination of lafutidine and its impurities in bulk and pharmaceutical dosage form

Abstract Background A simple and rapid stability-indicating, reversed phase ultra-performance liquid chromatography (UPLC) method was developed for the quantitative determination of lafutidine and its four potential impurities. Results Separation was achieved on Acquity BEH-shield RP18 UPLC column (3.0 mm × 100 mm, 1.7 μm) under the gradient mode of elution by using mobile phase A (0.02M diammonium hydrogen phosphate/acetonitrile, 80:20 v/v) and mobile phase B (0.02M diammonium hydrogen phosphate/acetonitrile, 30:70 v/v). The flow rate was maintained at 0.5 mL min−1. UV detection was carried out at 276 nm. Conclusions Stability-indicating capability of the developed method is established by analyzing forced degradation samples in which the spectral purity of lafutidine is ascertained along with the separation of degradation products from analyte peak. The developed UPLC method is validated as per International Conference on Harmonization guidelines with respect to system suitability, specificity, precision, sensitivity, accuracy, linearity, and robustness.

Fast and direct quantification of underivatized muscone by ultra performance liquid chromatography coupled with evaporative light scattering detection

A new reversed phase ultra performance liquid chromatography coupled with evaporative light scattering detection is developed for the fast and direct quantification of underivatized muscone in precious herbal medicine musk. Separation of muscone was achieved on a Waters Acquity BEH C18 (50 × 2.1 mm id, 1.7 μm) column. The runtime was as short as 5 min. The mode of evaporative light scattering detection was set at Impact On. The influence of evaporative light scattering detection condition on sensitivity was investigated. The optimized condition was: drift tube temperature at 30°C, gas flow rate 4.2 L/min. The method was validated with respect to the precision, sensitivity, accuracy, linearity, stability, and robustness were measured in this paper. The calibration curves showed good linear regression (r = 0.9914) within the test range. The recovery rate was 98.6%. The limit of detection for muscone was 2.0 ng. The validated method was rapid, simple, reproducible, and convenient for the quantification of muscone in musk and the related products.

Pharmacoproteomic Study of the Natural Product Ebenfuran III in DU-145 Prostate Cancer Cells: The Quantitative and Temporal Interrogation of Chemically Induced Cell Death at the Protein Level

A naturally occurring benzofuran derivative, Ebenfuran III (Eb III), was investigated for its antiproliferative effects using the DU-145 prostate cell line. Eb III was isolated from Onobrychis ebenoides of the Leguminosae family, a plant endemic in Central and Southern Greece. We have previously reported that Eb III exerts significant cytotoxic effects on certain cancer cell lines. This effect is thought to occur via the isoprenyl moiety at the C-5 position of the molecule. The study aim was to gain a deeper understanding of the pharmacological effect of Eb III on DU-145 cell death at the translational level using a relative quantitative and temporal proteomics approach. Proteins extracted from the cell pellets were subjected to solution phase trypsin proteolysis followed by iTRAQ-labeling. The labeled tryptic peptide extracts were then fractionated using strong cation exchange chromatography and the fractions were analyzed by nanoflow reverse phase ultraperformance liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry analysis using a hybrid QqTOF platform. Using this approach, we compared the expression levels of 1360 proteins analyzed at ≤ 1% global protein false discovery rate (FDR), commonly present in untreated (control, vehicle only) and Eb III-treated cells at the different exposure time points. Through the iterative use of Ingenuity Pathway Analysis with hierarchical clustering of protein expression patterns, followed by bibliographic research, the temporal regulation of the Calpain-1, ERK2, PAR-4, RAB-7, and Bap31 proteins were identified as potential nodes of multipathway convergence to Eb III induced DU-145 cell death. These proteins were further verified with Western blot analysis. This gel-free, quantitative 2DLC-MS/MS proteomics method effectively captured novel modulated proteins in the DU-145 cell line as a response to Eb III treatment. This approach also provided greater insight to the multifocal and combinatorial signaling pathways implicated in Eb III-induced cell death.

Plasmodium falciparum Malaria Elicits Inflammatory Responses that Dysregulate Placental Amino Acid Transport

Placental malaria (PM) can lead to poor neonatal outcomes, including low birthweight due to fetal growth restriction (FGR), especially when associated with local inflammation (intervillositis or IV). The pathogenesis of PM-associated FGR is largely unknown, but in idiopathic FGR, impaired transplacental amino acid transport, especially through the system A group of amino acid transporters, has been implicated. We hypothesized that PM-associated FGR could result from impairment of transplacental amino acid transport triggered by IV. In a cohort of Malawian women and their infants, the expression and activity of system A (measured by Na+-dependent 14C-MeAIB uptake) were reduced in PM, especially when associated with IV, compared to uninfected placentas. In an in vitro model of PM with IV, placental cells exposed to monocyte/infected erythrocytes conditioned medium showed decreased system A activity. Amino acid concentrations analyzed by reversed phase ultra performance liquid chromatography in paired maternal and cord plasmas revealed specific alterations of amino acid transport by PM, especially with IV. Overall, our data suggest that the fetoplacental unit responds to PM by altering its placental amino acid transport to maintain adequate fetal growth. However, IV more profoundly compromises placental amino acid transport function, leading to FGR. Our study offers the first pathogenetic explanation for FGR in PM.

Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography–tandem mass spectrometry

An ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid-liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7μm), a flow rate of 0.5mL/min, and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode, using two transitions for the compounds and one transition for the IS. The run time of the method was 8min including equilibration time. The calibration curves had R(2) values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0nmol/L to 0.10μmol/L (1.3-26ng/mL) and the limits of detections (LODs) from 1.0 to 20nmol/L (0.24-5.3ng/mL). LLOQ corresponds to 0.28-5.5pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC-MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC-MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.

Simultaneous determination of six estrogens in different water bodies by solid phase extraction and ultra performance liquid chromatography coupled with triple quadrupole mass spectrometry

A method has been developed for the simultaneous determination of six estrogens: (estriol, 17-beta-estradiol, 17-alpha-estradiol, estrone, ethinyloestradiol, diethylstilbestrol) in water environment by solid phase extraction and ultra performance liquid chromatography couple with triple quadrupole mass spectrometry. After enriched and purified by an HLB solid phase extraction column, the six estrogens were separated by a BEH C18 column and then determined by MS/MS with multiple reaction monitoring (MRM) mode. The quantification was achieved by internal standard method, with estriol-D3, estradiol-D2 and diethylstilbestrol-D8 as the internal standards. Good linear relationships were obtained in the mass concentration range of 1.0-100 microg/L with high correlation coefficients over 0. 998 2. The limits of detection and quantification for the six estrogens were 0.27-0.45 ng/L and 1.08-1.78 ng/L, respectively. The recoveries were in the range of 68.3%-97.4% at the three spiked levels and with the relative standard deviations below 15%. The method shows high sensitivity, good precision, extensive analytical range and high analytical speed. It was successfully applied to the simultaneous detection of the six estrogens in four different water bodies including surface water, waste water, source water of drinking and domestic water.

Separation and quantification of cysteine, glutathione and phytochelatins in rice (Oryza sativa L.) upon cadmium exposure using reverse phase ultra performance liquid chromatography (RP-UPLC) with fluorescence detection

Thiol compounds in plants, such as cysteine (Cys), glutathione (GSH) and phytochelatins (PCs), may be induced upon exposure to excessive potentially toxic metals and metalloids. In this paper, Cys, GSH, PC2, PC3 and PC4 in rice leaf, stem and root samples were derived with monobromobimane (mBBr), separated by reverse phase-ultra performance liquid chromatography (RP-UPLC) and detected by fluorescence. The results showed that, after reduction with tris(β-chloroethyl)phosphate (TCEP), the above thiols were rapidly separated with binary gradient elution in 15 min. The calibration curve was linear in a range of 1–20 μg mL−1. The limit of detection (LOD) for thiols was 0.14 pmol for 1 μL injection (0.10 μg g−1 F.W), and the recovery was 91.55% on average. Compared with the previously reported methods, the technique developed in this work demonstrated higher speed and sensitivity in the simultaneous measurements of Cys, GSH and PCs in rice. Upon exposure of 1 μM Cd for 7 days, the PC2–4 were remarkably induced in rice roots, whereas the amounts of Cys and GSH, the dominant thiol compounds in rice leaves and stems, were reduced.

Isolation and Structural Elucidation of Novel Isomeric Process Related Impurities of Zolmitriptan

Four isomeric unknown impurities ranging from 0.08-0.12% were found in the purified sample of Zolmitriptan during the batch analysis by gradient reverse phase ultra performance liquid chromatography (UPLC) and their molecular weights determined by liquid chromatography mass spectroscopy (LC-MS) analysis. Subsequently, all the four impurities were isolated by flash chromatography followed by semi-preparative HPLC and characterized by 1H NMR, 13C NMR, 1H-1H COSY, HMBC, HSQC, MS spectroscopy and HPLC. The structures for these four impurities were assigned to be following Isomeric Impurity-1: 4-((3-(2-(dimethylamino)ethyl)-4-(2-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl) oxazolidin-2-one, Isomeric Impurity-2: 4-((3-(2-(dimethylamino)ethyl)-2-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl) oxazolidin-2-one-, Isomeric Impurity-3: 4-((3-(2-(dimethylamino)ethyl)-7-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl) oxazolidin-2-one, Isomeric Impurity-4: 4-((3-(2-(dimethylamino)ethyl)-6-(4-((oxazolidin-4-yl)methyl)phenyl)-1H-indol-5-yl)methyl) oxazolidin-2-one Isolation and characterization of impurities has helped us in improving the purity of API by removing these impurities using crystallization.

Influence of Nonpolar Substances on the Extraction Efficiency of Six Alkaloids in Zoagumhwan Investigated by Ultra Performance Liquid Chromatography and Photodiode Array Detection

A reverse phase ultra performance liquid chromatography and photodiode array (UPLC-PDA) detection method was established for the determination of six alkaloids in Zoagumhwan (ZGW), and further for investigating the influence of nonpolar substances on the extraction efficiency of these alkaloids. The method was based on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) column and mobile phase of aqueous phosphoric acid and acetonitrile including 0.05% buffer solution under gradient elution. ZGW samples of ZGW I, II, III and IV were obtained and prepared by pre-processing the crude materials of Coptidis rhizoma and Evodiae fructus using four technologies, namely direct water decoction, removal of nonpolar substances in Evodiae fructus by supercritical fluid extraction (SFE), removal of nonpolar substances in ZGW by SFE and removal of nonpolar substances in ZGW by steam distillation. The developed and validated UPLC-PDA method was precise, accurate and sensitive enough based on the facts that the six alkaloids showed good regression (r > 0.9998), the limit of detections and quantifications for six alkaloids were less than 28.8 and 94.5 ng/mL, respectively, and the recovery was in the range of 98.56%–103.24%. The sequence of the total contents of six alkaloids in these samples was ZGW II > ZGW IV > ZGW III > ZGW I. ZGW II, in which nonpolar substances, including essential oils, were firstly removed from Evodiae fructus by SFE, had the highest content of the total alkaloids, indicating that extraction efficiency of the total alkaloids could be remarkably increased after Evodiae fructus being extracted by SFE.

A simple and sensitive RP-UPLC method for the determination of 2-(4', 4'- Dibromomethylphenyl) benzonitrile impurity content in Irbesartan drug substance

This paper describes the development and validation of a simple reverse phase ultra performance liquid chromatography (RP-UPLC) method for the quantitative determination of 2-(4',4'-Dibromomethylphenyl)benzonitrile impurity content at low level in Irbesartan drug substance. Separation was achieved with 100mm x 2.1mm, 1.7μm particle size, ODS column. The mobile was a gradient prepared by simple phosphoric acid buffer, and Acetonitirle at a flow rate of 0.1 mL min-1, UV detection was performed at 205nm. The method was validated to confirm selectivity, precision, linearity and accuracy parameters as per ICH guideline. Because of its speed and accuracy the detection limit and quantification limit are found to be 0.41 μg ml-1 and 1.46 μg ml-1 respectively. Repeatability is good, with a relative standard deviation of 2.1% to 3.2%. The drug substance was subjected to stability studies as well as stress condition of hydrolysis, oxidation, photolytic, thermal and humidity degradation using optimized method condition to enhance low level of detection within the minimum acquisition time indicating the accuracy of the optimized RP-UPLC method. This simple, efficient methodology can be used for quality control bulk manufacturing as well as routine analysis.