C18 HPLC Research Articles

Determination of Menbutone Residues in Edible Swine Tissues Based on Solid-Phase Extraction and RP-HPLC

Background: Menbutone is widely used as a veterinary choleretic drug in many countries. There was no publicly available analysis method for the determination of menbutone residues in swine tissues. It is necessary to establish a method to control the maximum residue limit and ensure food safety of the public. Objective: The aim of this study is to establish an analytical method for the simultaneous determination of menbutone in muscle, fat, liver and kidney tissues from swine. Methods: MBT residue was extracted by acetonitrile from the tissues then purified by using a C18 solid phase extraction (SPE) cartridge and an alkaline alumina (ALA) SPE cartridge. MBT was detected by RP-HPLC and separation was achieved on a Shim-pack VP-ODS C18 HPLC column using phosphoric acid solution (0.5%, v/v) and acetonitrile (45/55, v/v) at a flow rate of 1.0 mL/min. The effluent was monitored at 235 nm, and the column temperature was set to 30°C. Results: MBT eluted at 6.3 min and no interfering peak nearby was observed. This linearity within the concentration range of 0.02 (LOQ) and 12 µg/mL (r2>0.9999, n=6). The accuracy ranged from 74.07 to 110.83% of the actual values. Intra and inter-day precision were within 15.11%. In the application study, MBT was detectable in continuously dosing MBT 10 µg/g/day to healthy swine for 7 days. Conclusion: The proposed method has specificity, accuracy, and sensitivity, with an excellent linear relationship that successfully applied to swine tissues.

Stability Testing of Compounding Capsule Combination between Paracetamol and Tramadol in a Private Hospital Semarang

A combination of Paracetamol and Tramadol is used in mild to severe pain management. In a private Hospital Semarang, this combination is included in fast-moving drugs that it is frequently compounded and prepared in advance. The study aims t o determine Beyond Use Date (BUD) in compounding capsules combination between Paracetamol and Tramadol samples. Beyond Use Date is a time limit indicating that a medicine beyond the expiration date must not be used and determined based on the results of stability testing. Samples were stored for 14 days in a tightly closed container far from direct sunlight in room temperature without AC (28 0 C) and without silica gel, room temperature with AC (25 0 C) without silica gel, and room temperature with AC (25 0 C) with silica gel. Furthermore, samples underwent physical and chemical stability testing. Physical stability testing was conducted using organoleptic testing by observing direct changes from the powder color in capsules, capsules form, and scent on the samples. Meanwhile, chemical stability testing was conducted by determining the content of active ingredient from the samples using reversed-phase HPLC method and C18 as the stationary phase, methanol and aquabidest (40:60) as the mobile phase, wavelength 271 nm, flow rate 0.6 mL/min and injection volume 10 μL. The result shows that s amples were physically stable for being able to retain the original physical properties showed by consistent powder color and capsules form, and there was no available scent. However, the chemical stability testing method was unable to separate and quantify the content of Paracetamol, Tramadol, and formed degradation products. It can be concluded that Beyond Use Date based on organoleptic and chemical stability testing for 14 days of compounding capsules combination between Paracetamol and Tramadol could not be determined. Nevertheless, this study showed favorable storage conditions in a tightly closed container far from direct sunlight in Room Temperature with AC (25 0 C) with Silica Gel.

Development of a RP-HPLC method for analysis of docetaxel in tumor-bearing mice plasma and tissues following injection of docetaxel-loaded pH responsive targeting polymeric micelles.

Background and purpose:A simple, rapid, and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method based on liquid-liquid extraction was developed and validated for determination of docetaxel (DTX) in plasma and homogenate tissues of tumor-bearing mice.Experimental approach:Samples were spiked with celecoxib as the internal standard and separation was achieved on a μ-Bondapak C18 HPLC column. The mobile phase consisted of a mixture of acetonitrile/water (40/60 v/v) at flow rate of 1.2 mL/min and the effluent was monitored at 230 nm.Results:Calibration curves were linear over the concentration range of 0.1-10 μg/mL of DTX in plasma and 0.25-50 μg/mL in tissue homogenates with acceptable precision and accuracy. The mean recoveries of the drug from plasma extraction was 94.6 ± 1.44% while those of tissue homogenates ranged from 73.5 ± 3.2 to 85.3 ± 2.8% depending on the type of tissues examined. DTX was stable in biological samples with no evidence of degradation during 3 freeze-thaw cycles and two months of storage at -70 ± 15 °C. The developed HPLC method was applied to quantify DTX in the mouse plasma and tissues after intravenous administration of 7.5 mg equivalent DTX/kg dose of DTX-loaded folic acid-polyethylene glycol-heparin-tocopherol (FA-PEG-HEP-CA-TOC) micelle formulation to female Balb/c mice.Conclusion:A simple, sensitive, rapid, accurate, and prudent RP-HPLC method was developed, validated, and applied for DTX determination in plasma and tissues.

DEVELOPMENT, VALIDATION OF RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF TENOFOVIR DISPROXIL FUMARATE, EMTRICITABINE AND RILPIVIRINE HYDROCHLORIDE IN BULK, FORMULATION AND USED IN NANOSUSPENSION

The present research work illustrates the development and validation of RP HPLC method for simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride in bulk and formulated in a pharmaceutical dosage form as a nanosupension. Antiretroviral drug treatment is the primary line of therapy for treating HIV. The multicomponent system formulated as a nanosuspension evidenced increased hydrophilicity, potency and decreased side effects. The separation was carried out by using efficient BDS hypersil C18 HPLC column with empower software. Combination method of Precipitation—ultrasonic homogenization was used for the preparation of the nanosuspension. The mobile phase used was methanol, water, acetonitrile (80:13.4:6.6) v/v and flow rate 1mL /min. The developed method was thus validated as per ICH guidelines for various parameters whose results advocated the reliability of the method. The results for parameters viz. retention times of tenofovir disproxil fumarate, emtricitabine and rilpivirine were 3.09 min, 2.78 min and 3.68 min, linearity range was between 7.5-90, 5-60, 0.625-7.5µg/mL, respectively. Thus the new RP-HPLC method is optimum, reliable and can be used for the simultaneous estimation of tenofovir disproxil fumarate, emtricitabine and rilpivirine hydrochloride.

Extraction Strategies for Simultaneous Determination of Florfenicol and Florfenicol Amine in Tilapia (Oreochromis niloticus) Muscle: Quantification by LC-MS/MS

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of the veterinary drug florfenicol (FF) and its major metabolite, florfenicol amine (FFA), in tilapia muscles (Oreochromis niloticus). Three different sample preparation procedures (DLLME, sub-zero, and modified QuEChERS) were tested. The best extraction results were obtained by using the modified QuEChERS. The quantification was made by using a LC-MS/MS analysis, with a Lichrocart Cartridge Purospher Star C8 HPLC column (250 mm×4.6 mm, 5 μm particle size). Analytes were separated with a mobile phase consisting of Milli-Q water to acetonitrile 40:60 (v/v), both with 0.1% formic acid. The validation parameters were recovery of 70 to 79% and 62 to 69%, limit of detection of 0.0625 μg g−1 and 0.125 μg g−1, and limit of quantification of 0.125 μg g−1 and 0.25 μg g−1, for FF and FFA, respectively. CCα was 1183 μg kg−1 and CCβ was 1365 μg kg−1 for FF, intraday and interday precision has CV ≤20%, and linear range was 0.625 to 5.00 μg g−1. This method was shown to be simple and rapid when compared to other, more conventional methods. Also, it has low reagent and solvent consumption, with low waste generation, which is in line with the principles of green chemistry. The method was successfully applied for the analyzes of tilapia exposed to the antibiotic.

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis, Multilaboratory Testing Study: Final Action 2017.03

Abstract Background: A multi-laboratory study was completed with AOAC First Action Method 2017.03, HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis. Objective: Ten laboratories from seven countries participated in the multi-laboratory study. Each laboratory analyzed 14 infant, pediatric, and adult nutritionals in duplicate. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk-based infant formula ready-to-feed (RTF) liquids, adult low-fat powders, and adult high-fat and high-protein ready-to-drink nutritionals. Methods: Tryptophan was released from the intact protein in product matrices with a combination of proteolytic enzymes found in pronase. Following proteolysis, tryptophan was quantitated by reverse-phase isocratic HPLC and fluorescence detection. Prepared samples were injected onto a C8 HPLC column with a methanol/0.05 M phosphate buffer mobile phase. Results: Overall for tryptophan, repeatability averaged 2.1% relative SD (RSD) with a range of 0.9–3.6% RSD, and reproducibility averaged 4.2% RSD with a range of 3.0–9.9% RSD. Conclusions: Repeatability Standard Method Performance Requirements (SMPRs®) were met for 13 of the 14 matrices and reproducibility SMPRs were met for 11 of the 14 product matrices analyzed.

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis, Multilaboratory Testing Study: Final Action 2017.03.

Background: A multi-laboratory study was completed with AOAC First Action Method 2017.03, HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis. Objective: Ten laboratories from seven countries participated in the multi-laboratory study. Each laboratory analyzed 14 infant, pediatric, and adult nutritionals in duplicate. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk-based infant formula ready-to-feed (RTF) liquids, adult low-fat powders, and adult high-fat and high-protein ready-to-drink nutritionals. Methods: Tryptophan was released from the intact protein in product matrices with a combination of proteolytic enzymes found in pronase. Following proteolysis, tryptophan was quantitated by reverse-phase isocratic HPLC and fluorescence detection. Prepared samples were injected onto a C8 HPLC column with a methanol/0.05 M phosphate buffer mobile phase. Results: Overall for tryptophan, repeatability averaged 2.1% relative SD (RSD) with a range of 0.9-3.6% RSD, and reproducibility averaged 4.2% RSD with a range of 3.0-9.9% RSD. Conclusions: Repeatability Standard Method Performance Requirements (SMPRs®) were met for 13 of the 14 matrices and reproducibility SMPRs were met for 11 of the 14 product matrices analyzed.

QbD-Driven Analytical Method Development and Validation for Raloxifene Hydrochloride in Pure Drug and Solid Oral Dosage Form

The present research work encompasses systematic development of a simple, fast, sensi- tive, reproducible and cost-effective reversed-phase high-performance liquid chromatographic (RP-HPLC) method, applying the principles of Quality by design (QbD) for quantification of raloxifene hydrochloride (RLX). Reversed-phase chromatography was carried out using Waters Acquity high-performance liquid chro- matographic system (HPLC), equipped with photodiode array (PDA) detector controlled by Empower 2® software, using a Hibar Purospher® STAR C18 (250 x 4.6 mm; 5 μm) HPLC column. Initial risk assessment followed by screening studies was carried out using a four-factor-eight-run fractional factorial design (FFD). Subsequently, optimization studies were carried out employing a central composite design (CCD) with flow rate and mobile phase ratio as the critical method parameters (CMPs), and tailing factor (TF), % assay and theoretical plate count (TPC) as the critical analytical attributes (CAAs). The optimal chromatographic sepa- ration was conducted using graphical and numerical optimization with the mobile phase composition of methanol and sodium acetate buffer (pH 4) in the ratio of 50:50 (% v/v) at a flow rate of 1mL/min, an oven temperature of 40°C at a λmax of 287 nm. Linearity was observed in the concentration range of 0.5-100 μg/mL, with a correlation coefficient of 0.999. Further, the LOD and LOQ were found to be 0.5432 ng/mL and 1.6461 ng/mL respectively. The developed method was validated as per the ICH Q2 (R1) guidelines, and the principles and science of analytical quality by design (AQbD) for establishing a high degree of linearity, accuracy, precision, sensitivity and robustness, for estimating RLX in bulk drugs and marketed formulations.

Chemical Stability and Spectroscopic Characterization of Balofloxacin and its Stress Degradation Products by LC-PDA, LC-MS/MS, NMR and FT-IR

The present manuscript describes the preparative isolation and characterization of three degradation products of Balofloxacin (BFX). The drug is subjected to different stress conditions under acidic, basic, hydrolysis, UV, thermal and oxidative stress conditions, three degradation products (DP-1 to DP-3) were formed under acidic, basic, oxidative stress conditions and no considerable degradation was observed in hydrolytic, thermal, UV stress conditions. The degradants were adequately separated by using stationary phase of LiChrospher® RP-18 HPLC C18 (250 mm × 4.6 mm i.d., 5 μm) column with mobile phase consisting of 20 mM aqueous ammonium acetate: acetonitrile: methanol (55:25:20, v/v/v) subsequently the degradation products (DP-1; m/z 194, DP-2; m/z 359, DP-3; m/z 365) were isolated by semi preparative chromatography and further characterized by LC-ESI-MS/MS, HRMS, NMR and FT-IR methods. On the evidence of spectral data three major DP’s were confirmed and the developed assay method was validated as per ICH Q2 (R1) guideline. Finally, the proposed degradation pathways were extenuated by LC-ESI-MS studies. The proposed analytical stability indicating assay method was successfully applied to bulk drugs and formulations.

New HPLC–MS method for rapid and simultaneous quantification of doxycycline, diethylcarbamazine and albendazole metabolites in rat plasma and organs after concomitant oral administration

A sensitive and relatively fast, cost-effective high-performance liquid chromatographic method coupled with mass spectrometer (HPLC-MS) is herein reported for the first time for a simultaneous quantification of plasma and organs concentration of three therapeutic agents that are widely used in treatment of lymphatic filariasis (LF), namely, doxycycline (DOX), diethylcarbamazine (DEC) and albendazole (ABZ) metabolites. The method was developed and validated as per ICH and FDA guidelines and successfully employed to quantify DOX, DEC and ABZ metabolites (albendazole sulfoxide (ABZ-OX) and albendazole sulfone (ABZ-ON)) in the plasma and organs of Sprague Dawley rats after oral concomitant administration of the above mentioned therapeutic agents. Importantly, a simple, one-step protein precipitation and extraction method was used to extract the four compounds efficiently with a recovery in the range of 79.88 ± 5.02%–90.71 ± 5.13%, 85.72 ± 7.22%–93.17 ± 5.55%, 94.38 ± 7.35%–101.00 ± 8.88% and 94.38 ± 7.35%–99.87 ± 10.22% in plasma and organs for DOX, DEC, ABZ-OZ and ABZ-ON, respectively. Separation of all analytes was performed on a Xselect CSH™ C18 HPLC column (Waters, 3.0 x 150 mm, 3.5 μm particle size) with gradient elution employing a mobile phase consisting of 0.1% v/v formic acid in water and methanol with a run time of 20 min. Quantification was carried out employing a single, quadruple MS detector operated with single ion monitoring (SIM) mode and the ion transitions at m/z of 445.4, 200.2, 282.3 and 298.3 for DOX, DEC, ABZ-OX and ABZ-ON respectively. The MS response for plasma samples was linear across the concentration range of 2.5–2500 ng/mL for DOX, 0.5–500 ng/mL for DEC, 1–1000 ng/mL for ABZ-OX and ABZ-ON with a correlation coefficient (r2) ≥ 0.998. The method was selective, precise and accurate. This method allowed us to get an insight into the pharmacokinetics and biodistribution of the three therapeutic agents after simultaneous oral administration to Sprague Dawley rats. This bioanalytical method could provide a reliable, reproducible and excellent tool for routine therapeutic drug monitoring of the above mentioned therapeutic agents and also support other clinical pharmacokinetic-based studies.

Development and Validation of A Fast, Simple And Specific Stability Indicating RP-HPLC Method for Determination of Dexpanthenol in Eye Gel Formulation.

In this study a simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of Dexpanthenol in an artificial tear formulation. The chromatographic separation was performed on a HPLC C18 column (25.0 cm ´4.6 mm, 5 mm) using a mixture of 0.037 M monobasic potassium phosphate in water (adjusted with 0.1% (v/v) phosphoric acid to a pH of 3.2) and methanol (90:10). The flow rate was set at 1.5 mL/min and Dexpanthenol concentration was determined at λmax = 205 nm. The HPLC analysis method was validated in terms of linearity, precision, accuracy, specificity, and sensitivity according to International Conference on Harmonization (ICH) guidelines. The results indicated that the retention time was 8 min and no interferences were observed from the formulation excipients and stress degradation products. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 10 - 100 μg/mL; the regression coefficient was 0.996 and the linear regression equation was y = 20.011 x + 146.83. This HPLC method was precise and accurate in the range of 10 – 100 μg/mL. Also, the dexpanthenol concentration in artificial tear formulation was determined by this HPLC method, which was in accordance with the label claimed. This validated HPLC method could be used for routine analysis, quality control and the stability of analysis of eye gel containing dexpanthenol formulations.

The plasma peptidome

BackgroundIt may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.MethodsA systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.ResultsPeptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.ConclusionsThe random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

Nonhydroxylated 1-O-acylceramides in vernix caseosa

Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1-O-acylceramides (1-O-ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-O) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1-O-ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1-O position, 24 or 26 carbons in the N position, and sphingosine. The 1-O-ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1-O-ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1-O-ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF DABIGATRAN ETEXILATE RELATED SUBSTANCE IN PHARMACEUTICAL DOSAGE FORM BY REVERSE‑PHASE – HIGH‑PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The objective of the study was to develop and validate new, simple, and selective reverse-phase–high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of Dabigatran Etexilate (DE) and its impurities in pharmaceutical dosage form as per the International Conference on Harmonization guidelines.Method: Chromatographic analysis was performed on Princeton SPHER-l00 C18 (250 × 4.6 mm, 5 μm) HPLC column, maintained at 50°C column temperatures, 6°C sample tray temperature, and detection monitored at 225 nm. The mobile phase consisted of acetonitrile:phosphate buffer (pH 2.5) (33:67 V/V). The flow rate was maintained at 1.0 ml/min.Results: The system suitability results indicate good performance of the system. Specificity study indicates that there is no interference of placebo and blank. The percentage relative standard deviation (RSD) of six preparations for known and unknown impurity in the sample solution is found below 10%; hence, the method is precise. The calibration curve for DE (unknown impurity), Impurity A was linear from 0.38 to 4.5 μg/ml (correlation coefficients [r2] for unknown Impurity [DE] and Impurity A are 0.996 and 0.999, respectively). The calibration curve for Impurity B and Impurity E was linear from 0.38 to 9.00 μg/ml (r2 for Impurity B and Impurity E are 0.999 and 0.999, respectively); hence, the method is linear. Accuracy for DE (unknown Impurity), Impurity A, Impurity B, and Impurity E are found within 80%–120%; hence, the method is accurate. The percentage RSD for a standard solution is found below 5% up to 24 h, and percentage impurity change in the sample solution is found below 0.1% up to 18 h; hence, standard solution is stable up to 24 h, and sample solution is stable up to 18 h.Conclusion: The developed method is new, simple, adequate, specific, precise, linear, and accurate for the determination of DE and its impurities in pharmaceutical dosage forms.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ASPIRIN, ROSUVASTATIN, CLOPIDOGREL IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: To develop a novel, accurate, precise and linear reverse phase high-performance liquid chromatography (RP-HPLC) and Stability Indicating Assay Method (SIAMs) for simultaneous, qualitative and quantitative estimation of aspirin, rosuvastatin and clopidogrel in bulk and pharmaceutical dosage form as per International Conference on Harmonization (ICH) guidelines.Method: In the present work, good chromatographic separation was achieved by isocratic method using a BISCOF HPLC C18 column (250 mm ×4.6, 5 µm) and a mobile phase consisting of water at pH 2.51 with 0.1 % (v/v) orthophosphoric acid (OPA): acetonitrile in the ratio 50:50, at a flow rate of 1 ml/min. The effluents obtained were monitored at 237 nm with the UV-visible detector.Results: The retention time of aspirin, rosuvastatin, and clopidogrel was found to be 4.3 min, 7.6 min and 16.6 min respectively. For linearity seven-point calibration curves were obtained in a concentration range from 1-7 µg/ml for aspirin, rosuvastatin and clopidogrel with correlation coefficient 0.999, 0.9989, 0.9988 respectively. The high recovery values (99%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise. In the present study stability indicating an RP-HPLC method for the combination was tested by degrading the drugs together under various stress condition like acid, base and neutral hydrolysis, oxidation, thermal and photolytic stress which is recommended by ICH.Conclusion: The developed RP-HPLC method is simple, economic, specific, accurate and precise for the simultaneous estimation of aspirin, rosuvastatin, and clopidogrel in the combined capsule dosage form. The developed stability indicating analytical method can be used to check the stability of the compounds and was found suitable to determine % degradation of drugs in pharmaceutical dosage form.

Quantitation of plasma and urine 3-hydroxyglutaric acid, after separation from 2-hydroxyglutaric acid and other compounds of similar ion transition, by liquid chromatography-tandem mass spectrometry for the confirmation of glutaric aciduria type 1

BackgroundGlutaric aciduria type 1, a deficiency of glutaryl-CoA dehydrogenase, causes an accumulation of neurotoxic metabolites glutaric acid and 3-hydroxyglutaric acid (3-HGA). Testing of these analytes is routinely done by GC–MS but seldom account for interference from isomers or compounds with similar ion transitions. We developed a liquid chromatography tandem mass spectrometry method that accurately measures 3-HGA in urine and plasma specimens, while utilizing similar reagents and instrumentation used for the routine performance of amino acid and acylcarnitine analysis in determining the diagnosis of several metabolic disorders. MethodPlasma and urine samples were added aliquots of the deuterated 3-HGA internal standard and acetonitrile. The protein-free supernatant was brought to dryness, and the residue derivatized using 3 M HCL in 1-butanol with heating. The dried derivative was then reconstituted in 50% methanol-water solution and aliquot transferred to an HPLC vial for analysis by LC-MS/MS. Separation was performed using a C8 HPLC column under flow gradient conditions of 0.2% formic acid in water and methanol, respectively. Ionization was by ESI and detection of selected precursor-product ion transitions by multiple reaction monitoring (MRM) in positive mode. ResultsThe butyl-ester derivative of 3-HGA eluted at 7.82 min while 2-hydroxyglutaric acid (2-HGA) eluted at 8.21 min. This was equivalent to a separation factor of 1.05 and a resolution of 1.03, respectively. The 3-HGA calibration curve was linear over the range 6.20–319 ng mL−1 (r2 = 0.9996), and the reportable range determined by the linearity was found to be 1.54–384 ng mL−1. The calculated limits of detection and quantitation were 0.348 and 1.56 ng mL−1, respectively. Intra- and Inter-assay %CVs for quality control plasma and urine samples ranged from 2 to 18%, with recoveries of 66–115%. The method correlated to the gold standard GC–MS method for both serum (r2 ≥ 0.996) and urine analysis (r2 ≥ 0.949). The concentration of 3-HGA in normal, non-GA1 individuals was ≤25.2 ng mL−1 (in plasma) and ≤ 35.0 μmol mmol−1 of creatinine (in urine). ConclusionsThis LC-MS/MS method accurately quantified plasma and urine 3-HGA concentration after successful resolution from 2-HGA and other compounds with similar ion transitions. This method is suitable for confirmatory testing of 3-HGA, as a follow-up to an abnormal newborn screen test result, with concern for GA type 1.

Bioassay-Guided Fractionation of Wound Healing Active Compounds From Piper nigrum L. Berries Extract in Malaysia

The study aimed to further evaluate the wound healing property of P. nigrum L. using bioassay-guided fractionation method. The ethanolic extract of Piper nigrum L. was fractionated in two stages using column chromatography and preparative reversed phase C18 HPLC, respectively. Significant wound healing properties screened using the scratch wound assay was observed through the cell migration assay in fraction number three (PNE3) out of the 14 fractions, exhibiting 36.7% and 43.8% closure of wound gap within 20 hours at concentration of 0.3 and 1.0 µg/mL, respectively. Sub-fractions which were further fractionated from PNE3 showed comparatively reduced wound healing activity using the same bioassay. The sub-fractions were also compared to Piperine, a major component of P. nigrum L. and the results were comparable. This experimental study revealed that Piperine works together with other compounds in P. nigrum L. to improve wound healing as claimed by those home remedy.

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ELLAGIC ACID AND QUERCETIN IN AN AYURVEDIC FORMULATION

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and acetonitrile in the ratio 60:40, at flow rate of 1.2 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 255 nm with UV-visible detector.Results: The retention time of ellagic acid and quercetin were found to be 1.65 min and 2.94 min respectively. Linearity of ellagic acid and quercetin were tested in the range of 6-14 ppm and 3-11 ppm respectively. The correlation coefficient for ellagic acid and quercetin were 0.997 and 0.993 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulation containing these two markers.

A NOVEL REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF ELLAGIC ACID, QUERCETIN, AND PIPERINE IN AYURVEDIC FORMULATIONS

Objective: The objective of the study was to develop a novel, accurate, precise, and linear reverse-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of ellagic acid, quercetin, and piperine in different Ayurvedic formulations and validate as per the International Conference on Harmonization guidelines. Methods: In the present work, a good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6×250 mm, 5 μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and acetonitrile in the ratio 60:40, a flow rate of 1.2 ml/min and column temperature maintained at 35°C. The effluents obtained were monitored at 255 nm with ultraviolet-visible detector. Results: The retention time of ellagic acid, quercetin, and piperine was found to be 1.65 min, 2.94 min, and 14.57 min, respectively. The linearity of ellagic acid, quercetin, and piperine was tested in the range of 6–14 ppm, 3–11 ppm, and 3–13 ppm, respectively. The correlation coefficient for ellagic acid, quercetin, and piperine was found to be 0.997, 0.993, and 0.99, respectively. The high recovery values (98–102%) indicate a satisfactory accuracy. The low percent relative standard deviation values in the precision study reveal that the method is precise. Conclusion: The developed method is novel, simple, precise, rapid, accurate, and reproducible for simultaneous quantitative estimation of ellagic acid, quercetin, and piperine in Ayurvedic formulations. Hence, the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulations containing these three markers.

DEVELOPMENT AND VALIDATION OF A RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF QUERCETIN, ELLAGIC ACID AND RUTIN IN HYDROALCOHOLIC EXTRACT OF TRIPHALA CHURNA

Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of quercetin, ellagic acid and rutin in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines.Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3 with orthophosphoric acid) and methanol in the ratio 55:45, at flow rate of 1 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 254 nm with a UV-visible detector.Results: The retention time of quercetin, ellagic acid and rutin were found to be 7.52 min, 9.10 min and 12.47 min respectively. Linearity of quercetin, ellagic acid and rutin were found in the range of 8-12 ppm, 9-17 ppm and 7-11 ppm respectively. The correlation coefficient for quercetin, ellagic acid and rutin were 0.997, 0.999 and 0.999 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise.Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of quercetin, ellagic acid and rutin in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulation containing these three markers.