Dicloxacillin and Ampicillin, an Antibiotic Mixture Analyzed with HPLC – AppNote

Ampicillin and Dicloxacillin were retained with minimal amounts of buffer concentration and with good run-to-run precision in this HPLC method that can be readily transferred to LCMS. The combination of both drugs presents a mixture of polar and non-polar antibiotics that may incur difficulties to retain on conventional HPLC columns in Reversed Phase. This drug mixture […]

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Digoxin Analyzed with HPLC – AppNote

Forced Degradation Method Digoxin had several Sugar Residues that can be hydrolyzed in acidic conditions. In this Forced Degradation Method, six degradants are observed. Some of these Peaks are likely the various hydrolyzed forms, including the Aglycone Digoxigenin. The Method is capable of Separating a wide variety of Degradants. Figure A shows the Chromatogram of […]

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Diphenhydramine HCL Analyzed with LCMS – AppNote

Online SPE with a Simple Mobile Phase Step Gradient When a Mobile Phase containing 80% Solvent B (with 10mM Pyridine in Solvent A and B) is used Diphenhydramine Hydrochloride (DPH) is fully retained on the Column, meaning it does not elute and continues to be retained until it would overload the Column if injections were […]

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Diphenhydramine HCL Capsule Analyzed with HPLC – AppNote

Assay Method for Diphenhydramine HCL without Ion Pairing Agents Diphenhydramine has a tertiary amine functional group that can produce tailing with conventional Columns in Reversed Phase HPLC. The USP Method features a Triethylamine additive in the Mobile Phase for this reason. These additives often take a significant time to completely load onto the Column and […]

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Dipyridamole Analyzed with HPLC – AppNote

Several Tertiary Amines on a Molecule that is Easily Retained This Method for the Analysis of Dipyridamole USP Tablet Formulation is easy to do. Note the excellent Resolution from capsule excipients, along with the excellent Peak shape for this complex, difficult molecule.   Peaks: 1. Dipyridamole 2. Tablet Excipients Method Conditions Column: Cogent Bidentate C18™, 4μm, […]

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Docusate Sodium Analyzed with HPLC – AppNote

A Simple Gradient with Good Separation    The official USP assay method for Docusate Sodium tablets calls for a Mobile Phase of 50% 7mM Ammonium Acetate / 50% Acetonitrile at 40°C. However, retention for the API and the System Suitability compound Methyl Paraben were found to be low using these conditions. This Method not only […]

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Doxapram HCL Analyzed with HPLC – AppNote

Easy, Rapid Isocratic Assay of A Polar Compound Automating a Method is a distinct advantage over the official USP Method for Doxapram which is by Titration. Not only will this Method save time and money, but will reduce errors. Simple to perform, the Mobile Phase can be quickly prepared without errors. A highly Symmetrical Peak […]

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Doxycycline Analyzed with HPLC – AppNote

Forced Degradation Method Using Shape Selectivity Doxycycline is known to epimerize at the C4 and C6 positions in acid conditions. Epimers can be difficult to separate in Reversed Phase due to subtle differences in hydrophobicity. However, this Method differentiates based on shape as well as hydrophobicity and is therefore well suited to this Isomer separation. […]

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Doxycycline and Methacycline Analyzed with HPLC – AppNote

Simple Isocratic Method for API and Main Impurity Formulations of Doxycycline may contain a Methacycline Impurity.  As such, it is crucial for analytical methods to be able to separate these two peaks. Using this simple isocratic Method, the baseline resolution is obtained for this pair. Peaks: 1. Methacycline 2. Doxycycline * Impurities in doxycycline standard […]

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Doxylamine Succinate Tablet Analyzed with HPLC – AppNote

Shorter Run Time than USP Method The USP Assay Method for Doxylamine Succinate tablets uses Triethylamine and Sodium Lauryl Sulfate in the Mobile Phase. These reagents are slow to fully load onto the Column, resulting in long run times and poor Robustness. This Method uses Trifluoroacetic Acid to get an excellent Peak Shape. An overlay […]

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Dyclonine HCL Analyzed with HPLC – AppNote

A Reproducible Method for Detection of Dyclonine Hydrochloride A rapid, sensitive, and reproducible Method has been developed for detection of Dyclonine HCL. The data below, (overlay of 20 chromatograms ) illustrates how the compound can be adequately Retained and detected using HPLC, with both excellent Peak Shape and run-to-run precision. Using a simple Gradient in […]

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Effexor Capsule Analyzed with HPLC- AppNote

Reducing Tailing for Venlafaxine with HPLC The USP assay Method for Venlafaxine capsules uses Triethylamine and Phosphoric Acid in the Mobile Phase, both of which are incompatible with LC-MS. The system suitability for Venlafaxine tailing factor is 2.0, indicating the compound has a tendency for tailing. Here a sharp symmetrical peak is observed using Formic […]

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Ethambutol Analyzed with LCMS- AppNote

Retention of Hydrophilic Analyte in a Drug Product When Ethambutol is analyzed on many C18 Columns, it strongly interacts with residual Silanol groups in the Stationary Phase. These interactions normally cause excessive Retention and severe Peak broadening. In this LCMS Method, Formic Acid is all that is required for the analysis of charged drugs like […]

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Etodolac Tablet Analyzed with HPLC – AppNote

An Assay Method for Etodolac Tablets The USP Assay Method for  Etodolac tablets uses a Phosphoric Acid based Mobile Phase which is not LCMS compatible. The Method in this Application Note is more versatile since it can be used for HPLC or LCMS. The Gradient can be adjusted if lower Retention is required. Data from […]

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Famotidine Tablet Analyzed with HPLC – AppNote

API Separation from Matrix Component This Method for Analysis of Famotidine Tablets is easy to perform and produces a Symmetrical Peak Shape for the API. This compound has numerous amines which can be problematic in terms of Peak Shape with conventional Columns. Separation from a component from the tablet extract matrix is obtained as well, […]

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Fenfluramine & Phentermine Analyzed with HPLC – AppNote

Assay for Anti-Obesity Combination Capsule & Tablet This Application  Note illustrates Separation of similar compounds in the Phenethylamine class of compounds; Fenfluramine & Phentermine. These two compounds in particular were once used as APIs in the same formulation. The data shows how these Phenethylamines can be easily Retained and Separated with an Isocratic Mobile Phase […]

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Fenofibric Acid Capsule Analyzed with HPLC – AppNote

Simple Isocratic LCMS Compatible Assay Method The USP Assay Method for Fenofibric Acid uses a Phosphate buffer which is incompatible with MS detection. In this isocratic Method, only Formic Acid is needed in the Mobile Phase. The Peak Efficiency and Symmetry are excellent and the run-to-run precision is reliable and robust, as the 10 Chromatograms […]

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Fexofenadine Analyzed with HPLC – AppNote

Simple Isocratic Assay Method without Amine Additives Fexofenadine, antihistamine, has a number of aromatic moieties and the tertiary amine in its chemical structure that can cause Peak tailing issues when using most L11 Columns for Analysis. The USP Assay Monograph for Fexofenadine Tablets uses Triethylamine to reduce tailing. With this Method, Trifluoroacetic Acid is used […]

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Fioricet Analyzed with HPLC – AppNote

Gradient Separation of Acetaminophen, Caffeine, and Butalbital In this Method, a simple Reversed Phase gradient is used to separate the three components of a Fioricet tablet. Figure A shows the five-run overlay obtained from the gradient. Figure B shows a “zoomed in view” so that the Butalbital Peak can be seen clearly. Peaks: 1. Acetaminophen […]

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Fluconazole Tablet Analyzed with HPLC – AppNote

Assay Method for Amine Containing API This Application Note shows a simple Gradient Method for Assay of Fluconazole Tablets. In Reversed Phase HPLC, notable tailing was observed due to the two heterocyclic amine groups. Use of Aqueous Normal Phase (ANP) HPLC produced a sharp Peak for this API. Data from three different Column lots is […]

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Fluoxetine (Prozac®) – AppNote

Excellent Retention and Peak Shape with Amide Column Fluoxetine can have a tendency to tail in some HPLC methods due to its secondary amine group. However, peak shape with the Cogent Amide Column was found to be highly symmetrical without the aid of ion pairing agents. This allows the method conditions to be adapted to […]

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Fluoxetine Orthogonal Assay Method – AppNote

Simple Methods Without the Use of Ion-Pairing Agents Unlike the USP assay method, Ion Pair Agents are not needed in this application. Both figures show how good Peak symmetry can be achieved either in the Reversed Phase (RP, Figure A) or Aqueous Normal Phase (ANP, Figure B) mode with only Formic Acid as the Mobile […]

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Forced Degradation of Atorvastatin by LCMS – AppNote

Separation of API from its Lactone Degradation Product Atorvastatin is separated from its main Degradation product in this Method with a simple linear Reversed Phase Gradient. With the use of LCMS, the identity of the Degradant can be confirmed from its m/z value. The Degradation is an intramolecular Fischer esterification, which is catalyzed under Acidic […]

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Formoterol Fumarate Analyzed with HPLC – AppNote

Forced Degradation Methods  Forced degradation studies are useful for developing Stability Indicating Methods of Pharmaceuticals. In this Application Note Separation is obtained between the API and a Degradant formed under two different, strong acidic conditions. The non-degraded Sample produced a sharp, Symmetrical Peak for the API shown in Figure A. In Figure B, strong acid […]

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Furazolidone Analyzed with HPLC – AppNote

Improved Specificity Compared to USP Assay Method The USP Assay Method for Furazolidone is performed by UV Spectrophotometry. This HPLC Method provides more Robustness and Specificity for the analysis. Separation of Furazolidone from the structurally similar compound Nitrofurantoin is shown in the figure. Nitrofurantoin can be used as an internal standard to obtain more robust […]

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Furazolidone Analyzed with HPLC – AppNote

API and Related Compounds Separation Furazolidone was Separated from related compounds in a commercially available pharmaceutical preparation. Excellent Peak shape and Selectivity from the related compounds was achieved with this Robust Method making it a great choice for QC applications even when using aggressive Mobile Phases that include acids. Molecular weight of Furazolidone is 225.16. […]

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Furosemide Analyzed with HPLC – AppNote

Furosemide & Related Compound with Improved Peak Shapes Furosemide and its related compound can be a difficult molecule to chromatograph with conventional L1 (C18) Columns due to Silanol activity. With this Method, the Peak shape of Furosemide and its related Compound A is excellent and baseline Resolution is achieved between this specified impurity and Furosemide. […]

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Gabapentin Drug Product Analysis by LCMS – AppNote

Isocratic Retention with LCMS Gabapentin was retained successfully with a very symmetrical peak. Two different Mobile Phases were used and the advantage of the Mobile Phase used in Figure A is shown to be that Isopropanol present in the solvent A helps clean the Column which can be useful if biological samples containing lipids are […]

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Gentian Violet Analyzed with HPLC – AppNote

Separation of Related Compounds Gentian Violet degrades in solution, forming complex set of Degradation products. This Method can easily separate out the various compounds formed in Solution. The figure below shows a USP Standard of Gentian Violet after stressing it in solution thus creating Degradation products. With excellent Selectivity well as great Peak shape this […]

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Glimepiride Tablet Analyzed with HPLC – AppNote

Isocratic Assay Method for a Diabetes Drug In this study, an Isocratic, HPLC Method for Glimepiride Analysis in a tablet formulation is presented. The USP Assay Method for this drug calls for a Phosphate Buffer and is therefore not LCMS compatible. This Method uses Formic Acid instead and produces a Symmetrical, well-Retained Peak. Therefore the […]

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Glipizide Analyzed with HPLC – AppNote

Separation of API from Acid Degradation Products This Method illustrates the capabilities to separate several Glipizide Degradants that are formed under acidic conditions. Good separation is obtained as well as Sharp, Symmetric Peak shapes for each compound. The Retention times show good repeatability as well, as demonstrated in the Figure overlays. Also, the Mobile Phase […]

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Glyburide and Metformin Analyzed with HPLC – AppNote

Separation of “Highly Polar” and “Non-Polar” Compounds in one Isocratic run In this Method, the polar compound, Metformin, and the nonpolar compound Glyburide, can be retained on the same Stationary Phase (see A & B). Depending on the Mobile Phase composition either Metformin or Glyburide can be retained longer. Method Conditions Column: Cogent Bidentate C18™, […]

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Glycolic Acid Analyzed with HPLC – AppNote

Analysis in Urine Samples An enzymatic Method is widely used for the determinations of Glycolic Acid in various products, but it suffers from instability and very high cost of the enzyme (Glycolate Oxidase). The LCMS analysis presented here is rapid, simple, selective, and suitable for routine analyses of urine samples. It offers numerous advantages including […]

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Guanfacine HCL and Degradants Analyzed with HPLC – AppNote

USP Method with an SDS Resistant Column The US Pharmacopeia (USP) lists several known degradation products for Guanfacine. The Figure below shows a Separation of a degraded Guanfacine Drug Substance. This Method produces very Efficient results but what is important to note is that the Column used is extremely stable under the very aggressive Mobile […]

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Hesperidin Analyzed with HPLC – AppNote

LC-MS compatible method This method for analysis of Hesperidin produces an excellent analyte peak and shows separation from what appears to be two impurity peaks. The gradient method is LC-MS compatible and can be applied to many types of Hesperidin-containing samples such as citrus fruit extracts. The overlay of five runs shown in the figure […]

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Hormone Replacement Capsule Analysis with HPLC – AppNote

Separation of Estriol, Estradiol, & Progesterone This gradient method features a separation of the three components of a hormone replacement formulation. Figure A shows a five run overlay of the formulation extract injections. Figure B shows a zoomed-in view so that the Estriol and Estradiol Peaks, which are present in much lower concentration than Progesterone, can […]

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Hormone Replacement HPLC Methods – Extended AppNote

Ethinylestradiol, Norethisterone Acetate, Estriol, Estradiol, Progesterone, Separation in Hormone Replacement Formulations. Often, these formulations have multiple active ingredients present, therefore HPLC is an appropriate technique in obtaining the necessary specificity for a Quantitative Assay. This Method can retain the Hormones by conventional Reversed Phase HPLC and can also differentiate among the compounds by shape. The […]

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Hormone Replacement Tablet Analyzed with HPLC – AppNote

Isocratic Separation of Ethinylestradiol and Norethisterone Acetate In this simple HPLC Method, two hormones from a tablet extract are separated. Both analytes are adequately retained while keeping the run time to 10 minutes. The USP Monograph Method for Assay of the two compounds is lengthy and complex, while this Method is rapid and easy to […]

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Hydrocodone with Acetaminophen Analyzed with HPLC – AppNote

Simple, Robust Assay Method Hydrocodone can yield poor Peak shapes in many conventional Reversed Phase C18 Methods due to its tertiary amine group. The USP Assay Method for Hydrocodone in combination with Acetaminophen uses Triethylamine as a Mobile Phase additive to improve the Peak shape. In this Method however, only Trifluoroacetic Acid is needed in […]

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Hydromorphone Tablet Analyzed with HPLC – AppNote

Alternative to USP Method for this Opioid The USP Method for Hydromorphone Tablet Assay calls for the use of Sodium Dodecyl Sulfate (SDS) in the Mobile Phase. In addition to not being LCMS compatible, adding it is time consuming to load and unload from the HPLC column. Often the columns are permanently altered and cannot […]

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Hydrophilic Vitamins Analyzed with LCMS – AppNote

Different Mobile Phase for LCMS from a UV Method was Needed Hydrophilic Vitamin analyses were developed using UV detection. However, when the same Method and Mobile Phase was tried with MS Detection, the Peak intensities were very low and Peak Shape for Pyridoxine was not acceptable (see Red Peaks in Figures below). The Mobile Phase […]

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Hydroxychloroquine Sulfate – AppNote

Hydroxychloroquine Sulfate presents a few Complexities to Routine Chromatographic Analysis. The Amine Groups also interact with lone-Silanols, causing Peak Tailing on regular Silica Columns. In typical Reversed Phased Modes, Buffers are needed in higher concentrations to aid in retention, as well as Ion Pair Reagents. These additives can hinder coupling in Mass Spectrometry and also […]

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Hydroxytryptophan (5-HTP) Analyzed with HPLC- AppNote

Separation from Vitamin B6 Pyridoxine (Vitamin B6) is often added to formulations of 5-Hydroxytryptophan (5-HTP), as it assists in the conversion of 5- HTP to Serotonin. Therefore, it is of interest to separate these two compounds for an HPLC assay Method of this combination formulation. The Method shown provides good resolution between the two peaks […]

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Ibuprofen Assay Method Reproducibility with HPLC – AppNote

Extremely Precise, Assay Method This Assay Method for a common Ibuprofen formulation demonstrates Reproducibility and Robustness as the Figure below shows an overlay of four Chromatograms with different Columns lots (%RSD < 1). An important aspect of Column Selection for a Method is that the Retention behavior is consistent across numerous manufacturing batches. This is […]

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Indomethacin Capsule Analyzed with HPLC – AppNote

Simple Assay Method  This method for analysis of Indomethacin capsules is easy to perform and uses an LC-MS compatible Mobile Phase. The API peak that was obtained shows excellent efficiency and the data is very reproducible, as illustrated by the overlay of five runs shown in the figure. Peak: Indomethacin Method Conditions Column: Cogent Bidentate […]

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Ketoconazole Analyzed with HPLC – AppNote

Lipophilic Ketoconazole Retained using Aqueous Normal Phase HPLC Ketoconazole is a significantly lipophilic molecule (log P ~ 3.8) and is not generally suitable to ordinary Methods based on Hydrophilic Retention (HILIC). However, in this Method it is well retained using Aqueous Normal Phase (Inverse Gradient), therefore it is an Orthogonal Assay Method. This can be […]

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Ketorolac Separation with LCMS – AppNote

Analgesic agent There is a need for analysis of the Active Pharmaceutical Ingredient (API) in Ketorolac Tablet formulations in order to evaluate two different extraction protocols. The use of Ethanol as the extraction solvent was explored initially but DI Water was observed to produce superior extraction efficiency. The chromatograms show how the compound can be […]

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Ketotifen Analyzed with HPLC – AppNote

API with a Tertiary Amine can have a Great Peak Shape The tertiary amine group of Ketotifen makes it a challenging compound to analyze in HPLC.  In addition to good peak shape for this API, another peak can be identified which is baseline resolved from the main peak of interest. This Method is suitable for […]

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Lamotrigine Analyzed with HPLC – AppNote

USP Assay Method for a Very Polar Compound The USP Assay Method for Lamotrigine uses a relatively low pH of 2.0. These conditions may promote hydrolysis of the bonded phase in many L1 Columns, but the unique chemistry of the Cogent Bidentate C18 Column (L1) is very rugged and shows no loss of Retention for […]

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Lansoprazole Capsule Analyzed with HPLC – AppNote

Separation from matrix peaks Lansoprazole is separated from several Matrix Components in this simple assay method. The Mobile Phase is LC-MS compatible so the method could be applied to more complex samples such as plasma. Five runs are shown in the figure to illustrate the repeatability of the data. Peak:Lansoprazole Method Conditions Column: Cogent Bidentate […]

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