1,4-Benzodiazepines in Urine Analyzed with ANP – AppNote

1,4-Benzodiazepines from Urine Samples were analyzed using a Cogent Diamond Hydride 2.o HPLC Column successfully after Solid Phase Extraction (SPE). Four available Compounds (shown below) were well retained and separated. The procedure could be used for determination of this class of compounds in urine samples and other body fluids. Peaks: 1. Temazepam 301.0739 m/z [M+H]+ […]

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3-Hydroxy-3-Methylglutaric acid, HMG in Urine Analyzed with HPLC – AppNote

Important Biomarker for Diabetes and HMG Disorder is well retained with HPLC. A selective, specific, and sensitive HPLC method with LCMS Analysis has been developed for the determination of 3-hydroxy-3-methylglutaric acid (a.k.a. Meglutol) in urine samples. The method can be also used in the analysis of plasma samples after precipitation of plasma proteins with Acetonitrile. […]

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3,3′-Diaminobenzidine Analyzed with LCMS – AppNote

Retention and Separation is Easy with this Method 3,3′-Diaminobenzidine (DAB) is a very challenging compound for analysis using HPLC with a UV detector. It is highly polar and hence difficult to retain when Reversed Phase HPLC Columns are used. Moreover, when there are a significant number of Silanol Groups present on the surface of the […]

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4-(Methylamino)butyric Acid Analyzed with HPLC- AppNote

A gamma-Aminobutyric Acid (GABA) Derivative  This challenging polar compound is easily retained in this simple Method where repeatability of the data is excellent with %RSD<0.1 for the Retention Times. Peak: 4-(Methylamino)butyric Acid Method Conditions: Column: Cogent Diamond Hydride™, 4μm, 100Å Catalog No.: 70000-15P-2 Dimensions: 4.6 x 150mm Mobile Phase: —A: DI Water with 0.1% Formic […]

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Acetone as the Organic Mobile Phase Component – AppNote

“Green” Substitute for Acetonitrile in LCMS Method of 17 Amino Acids Acetone has a high UV absorbance “cutoff” and therefore is generally unsuitable for HPLC with UV Detection. However, this is not a problem when using LCMS or Evaporative Light Scattering Detectors (ELSD) such as the Shimadzu Prominence or the Corona CAD. Acetone can be […]

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Acetyl-L-Carnitine in Plasma Analyzed with LCMS – AppNote

Spiked Plasma Samples with Acetyl-L-Carnitine The Method in this Application Note was designed to be suitable for the routine analysis of Plasma Samples obtained from animal and human Pharmacokinetic studies in which Acetyl-L-Carnitine (ALC) is administered. The calibration curve prepared for the Plasma Samples showed good Linearity (R2 = 0.999) and the Precision was good […]

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Acrylamide HPLC Analyzed with HPLC – AppNote

Easy & Precise Retention of a Very Polar Compound  Acrylamide can be difficult to retain with conventional Reversed Phase Methods due to its polar nature. With this Method however, Retention is readily achievable using a simple, Isocratic Mobile Phase. The overlay of two Chromatograms using two different lots of HPLC  Columns in the Figure below […]

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Alprazolam (Xanax®) HPLC Method Transferred – AppNote

Benefit of 2.2um Column Analyzing Alprazolam with HPLC In this AppNote, we show that a transfer from a 4µm to 2.2µm Column, higher efficiency is observed with the smaller particle size Column. This can be very useful for analyses requiring detection at low concentrations. The Peak shape is very sharp using the 2.2um Column (Figure. […]

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Alprazolam Analyzed with HPLC – AppNote

Robust Separation of API from USP Internal Standard The USP Assay Method for Alprazolam uses a bare Silica Column and a complex Mobile Phase consisting of Acetonitrile, Chloroform, Butyl Alcohol, and Acetic Acid. In this Method, a simple LCMS compatible Mobile Phase is used and produces excellent Peak shapes for both the API and its […]

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Amikacin & Tobramycin – AppNote

Highly Polar Antibiotic Separation These two Antibiotic agents present a considerable challenge to chromatographers. As highly hydrophilic compounds, they are generally poorly retained by conventional Reversed Phase methods that rely on an analytes hydrophobicity. In addition, they exhibit very low UV absorption and therefore more sophisticated detection methods are often required. In this application, an […]

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Amiloride and Triamterene in Urine – AppNote

LCMS Method for Spiked Urine Sample This application note describes the separation of two important  drugs for treatment of hypertension and congestive heart failure. Both compounds have numerous amine groups, which can contribute to Peak tailing in some instances. However, the Peak shapes observed here are both symmetrical and very efficient. This is accomplished using […]

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Amoxicillin Analyzed Orthogonally with HPLC – AppNote

Orthogonal Analysis to USP Assay Method for Validation The USP Assay Method for the antibiotic Amoxicillin is performed in Reversed Phase. Analysis by an Orthogonal Retention mode is very useful and can be readily done by Aqueous Normal Phase (ANP) mode of HPLC, using solvents common to both. A benefit of this Method is the […]

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Analysis of a Local Anesthetic Lidocaine, Aminoethylamide – AppNote

Method Development for a Local Anesthetic (A Case Study) An analyst was using a Cogent Diamond Hydride™ Column for evaluating Lidocaine methods and although they had good retention of the compound, they did not meet the required 0.3 – 0.5 AU when 0.1 mg/mL Lidocaine sample was injected for both 2μL and 10μL injection volumes. […]

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Analysis of Clopidogrel with LCMS – AppNote

Analysis of the API and Degradants A Sensitive, Selective, and rapid LCMS Method was developed for the simultaneous Quantification of Clopidogrel (Plavix®) and degradants. This Method can be used for the determination of Clopidogrel in commercial tablets for Quality Control with an application for content uniformity test. The Method is also stability-indicating as it is […]

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Analysis of Harmful Substances in Dietary Supplements – Extended AppNote

Dietary Supplements have had, in notable cases, ingredients added to them that have been implicated in serious health issues and even death. Two such pernicious compounds are Aegeline and 1,3-Dimethylamylamine. These two can be readily separated from the dietary supplement matrix using the Cogent Bidentate C18 2.o™   and Cogent Diamond Hydride™ Columns by LCMS […]

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Analysis of Nucleotides – AppNote

ATP & AMP Separated in Aqueous Normal Phase (ANP) The chromatogram below shows an example of the separation possible with this Method for three Adenosine analytes using an Aqueous Normal Phase (ANP) Gradient. Peaks: 1. Adenosine-3’,5’-Cyclic Monophosphate 2. Adenosine 5’-Monophosphate (AMP) 3. Adenosine 5’-Triphosphate (ATP) Method Conditions Column: Cogent Diamond Hydride™, 4μm, 100Å Catalog No.: […]

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Analysis of Thiopurines – AppNote

Separation using LCMS Gradient Method The Cogent Diamond Hydride Column with gradient elution was used for analysis of Thiopurines. Figure A shows the Peak of 6-TG, which also can be analyzed using Isocratic conditions. Good retention and symmetrical peak shape were obtained under the analysis conditions. Figure B represents two separated Thiopurines (Mono and Di- […]

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Anatoxin-a, ANTX-A Analyzed with LCMS – AppNote

Potent Neurotoxin Analyzed without Derivatization Anatoxin-a or ANTX-A is a low molecular weight compound with a secondary amine which can be difficult to retain with most Reversed Phase HPLC Columns. This Application Note illustrates a very simple Method for Retention of Anatoxin-a which does not require pre-Column or post Column Derivatization as with other Methods. […]

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Antibiotic Keflex Analyzed with HPLC – AppNote

Analysis of Polar Antibiotic Main Active Ingredient: Cephalexin Monohydrate Cephalexin Monohydrate, commercially known as Keflex, is a semi synthetic Cephalosporin antibacterial drug, analysis of which is of great interest to scientists from many research fields. There is a need for a rapid, efficient and inexpensive analytical method to be applied for analysis of formulations during […]

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Arginine, Lysine and Histidine Analyzed with LCMS – AppNote

Basic Amino Acids In Synthetic or Human Urine Can be Analyzed A “Cleanup” procedure for the isolation of the Basic Amino Acids in Urine was developed in this Method and no derivatization procedure was used. Three Basic Amino Acids were Separated using an inverse gradient or Aqueous Normal Phase (ANP) Chromatography. The advantages of this […]

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Aspartic Acid & Arginine Analyzed with LCMS- AppNote

Reduced Sample Prep and Handling Two amino acids are adequately Retained and can be easily quantified in this Method with very little Sample Prep. There is no need for any Pre-Column or Post Column derivatizing due to Mass Spectrometric Detection can detect these molecules and Aqueous Normal Phase (ANP) HPLC can Retain in their ionized […]

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Atenolol Tablet Analyzed with HPLC – AppNote

Quick Analysis without Ion Pair Agents The USP Assay Method for Atenolol uses Heptane Sulfonate and Dibutyl Amine in the Mobile Phase, which are slow to load and remove from a Reversed Phase HPLC Column. These reagents are added to improve the Peak Tailing, which must be not more than 2.0. The Method in this […]

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ATP, UDP-Glucose UDP-Galactose Separation from Red Blood Cells – AppNote

Separation of Nucleotides  In this application, we present an easy-to-use protocol for sensitive detection and quantification of nucleotides in red blood cell extracts.  Retention times were very reproducible with %RSD around 0.4. The desired separation between UDP-Glu and UDP-Gal was achieved, all other nucleotides eluted later and did not interfere with the analysis. ATP represents […]

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Avoid Sample Cleanup for Food and Beverages – Extended AppNote

3 Methods to Avoid Sample Cleanup with Histamine, Limonin & Folic Acid Food and beverage analysis is complicated by the complex nature of the sample. In many cases, extensive sample cleanup techniques such as Solid Phase Extraction (SPE) may be required to avoid problems associated with the sample matrix. In this Application Note, three strategies […]

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B Vitamins Analysis with HPLC – AppNote

Separation of Five Highly Polar Vitamins The B Vitamins are all highly polar and often ion pair agents are needed in Reverse Phase in order to retain them. However, these agents are incompatible with LCMS and therefore limit the applications of these methods. In this method using the Diamond Hydride Column, good separation is obtained […]

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Baclofen Analyzed with HPLC – AppNote

Very Polar API Retained with Precision This compound has two ionizable functional groups (an amine and a carboxylic acid), which makes it quite suitable for retention using this Method. A simple Gradient produced a very sharp Peak for this Analyte. These Method conditions can be applied to the routine Analysis of Baclofen Tablet formulations. Peak: […]

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Benzylpiperazine (BZP) – AppNote

Analysis in Hair Samples using LCMS An analytical protocol was developed for analysis of Benzylpiperazine in human hair samples. The elucidation of the chromatographic method was challenging due to the polar nature of BZP. The limits of detection / quantification for this method were determined to be 0.05 ng / mg for Benzylpiperazine in hair […]

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Bradykinin Analyzed with LCMS – AppNote

The Peptide Bradykinin Analyzed in Saliva Samples A simple, sensitive, and specific Gradient Method was developed for the Quantification of Bradykinin (BDK) in Saliva. In addition to the intact Peptide, the Gradient applied in the developed Method was designed to analyze its impurities and metabolites. Robustness of this Method, against small modifications in pH, flow […]

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Brazilian Tree Bark Analyzed with LCMS – AppNote

Identification of Quercetin, Rutin, and “Compound 1” in a Brazilian Tree Bark Over one quarter of natural medicines have been discovered in rainforests, and therefore analytical study of the indigenous flora and fauna is necessary. The Amazonian rainforest in Brazil is the most biodiverse region in the world. A Brazilian tree bark extract (Brownea Grandiceps) […]

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Caffeine in Coffee Analyzed with HPLC- AppNote

Unique Retention Mode Affords Superior Specificity Although Caffeine retains well in Reversed Phase, it is found to be difficult to obtain a well-resolved Peak free of interference from other matrix peaks with a complex sample such as coffee. In this Method, most of the matrix peaks elute near the void volume and do not interfere […]

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Cetirizine Tablet Analyzed with HPLC – AppNote

Efficient and Symmetrical Peak of API from Zyrtec® Tablet This Application Note shows how very Efficient Peaks can be achieved for Cetirizine from a tablet formulation and how the API Peak can be well Retained with excellent Symmetry. Even with ion pairing in Reversed Phase Methods, a Peak with acceptable Efficiency and Symmetry could be […]

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Cetylpyridinium Chloride Analyzed with HPLC – AppNote

Excellent Peak Shape for a Cationic Compound Commonly found in Household Products This Method demonstrates the potential to avoid Peak Tailing or Band Broadening with cationic compounds due to interaction with Silanols on the surface of Reversed Phase Column. Tailing can cause interference with Quantitation of a nearby Peak of interest as well. Since Cetylpyridinium […]

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Chenodeoxycholic Acid in Urine Analyzed with LCMS – AppNote

Chenodeoxycholic Acid (CDCA) Separation in LCMS The presence of an important bile acid, Chenodeoxycholic Acid, in human urine was detected using a simple Mobile Phase with the Method below. In addition to being simple and easy, it also provides a fast Equilibration between runs even while using a Gradient, for higher throughput. Peak: Chenodeoxycholic Acid […]

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Chlorhexidine Analyzed with HPLC – AppNote

Rapid, Reliable Assay without Ion Pairing Agents Chlorhexidine contains numerous amine functional groups that can have a detrimental effect on Peak Shape in HPLC. The USP Assay Method uses Triethylamine as an Ion Pairing Agent in the Mobile Phase for this reason. The Method developed in this Application Note uses only Formic Acid as the […]

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Chlormequat & Mepiquat Analyzed with LCMS – AppNote

Plant Growth Regulators CQ & MQ Retained and Separated Analysis of Chlormequat and Mepiquat can be challenging since they are extremely hydrophilic and are only weakly Retained in Reversed Phase HPLC which makes their Separation difficult. In addition, these compounds are Retained too strongly in Normal Phase (Non Polar Solvents) Chromatography. Due to this situation, […]

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Ciprofloxacin Analyzed With LCMS – AppNote

Retention with Good Peak Shape for this Polar Compound This Method illustrates how the MS compatible HPLC UV Methods described in various application notes can be successfully adapted for LCMS. The Peak Shape for this polar compound meets the System Suitability specification for the USP Tailing Factor. Peak: Ciprofloxacin 332.1405 m/z (M+H)+ Method Conditions Column: […]

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Ciprofloxacin LC-MS Compatible Assay – AppNote

Antibiotic Tablet Ciprofloxacin has several amine groups and therefore is susceptible to peak tailing in Reversed Phase methods. The USP assay method for Ciprofloxacin tablets uses Triethylamine as an Ion Pair Agent to reduce tailing and in addition, Phosphoric Acid is included in the Mobile Phase. These conditions make the USP method incompatible with MS […]

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Citric, Aconitic & Maleic Acids Analyzed with LCMS – AppNote

Low Molecular Weight Organic Acids Low mass Acids were retained and Separated in this Method with the Aqueous Normal Phase mode of HPLC. The Method was able to resolve the three compounds in 10 minutes. Limits of Detection (LOD) for them ranged from 0.05 to 24 micromolar. Peaks: 1. Maleic Acid 115.0031 m/z (M-H)- 2. […]

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Clopidogrel Analyzed with HPLC – AppNote

LCMS Compatible Separation of API from Degradants Although Clopidogrel, commonly known as Plavix, retains well in Reversed Phase, its Degradants showed low Retention even at high water content. Therefore this Method the compounds are retained on the basis of polarity. This Method uses MS compatible Solvents and provides good Retention and separation between the API […]

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Cogent Diamond Hydride 2.o HPLC Column Retention Time Precision – AppNote

This Graph shows a Study Investigating the Day to Day Retention Time Precision with two test solutes on the Cogent Diamond Hydride 2.o™ HPLC Column. Peaks: Blue Trace: Phenylalanine Red Trace: Phenylglycine Many factors can affect Column Robustness and lifetime, so it is not possible to define specific values for those parameters. Mobile Phase: —A: DI […]

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Cyanuric Acid and Melamine Analyzed with LCMS – AppNote

Simple Gradient Method with MS Detection This Method by LCMS detection eliminates derivatization which may be necessary with GCMS Methods.  Reversed Phased Methods may run into poor separation of the small and very polar Melamine, resorting to ion pairing reagents to improve separation.  The use of these ion pairing reagents counterintuitively results in ionization suppression […]

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Cyclobenzaprine Retention with an Inverse Gradient – AppNote

ANP Retention of a Hydrophobic Compound  This Application Note shows an example of a hydrophobic analyte retention in Aqueous Normal Phase (ANP / HILIC Like Gradient) HPLC. Although Cyclobenzaprine has a logP of approximately 4.6–4.9, it is well retained with a Symmetrical Peak Shape. Data from two production lots of Columns is shown to illustrate […]

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Cycloserine LCMS Separation of Impurities and Degradants – AppNote

LCMS Separation of Impurities and Degradants While two major impurities observed in this Cycloserine method were not directly identified, several possibilities can be suggested based on their m/z values in the Mass Spectrum and their relationship to the Serine structure. Serine was also identified but in very low abundance and only after the samples were […]

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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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Dimethylolpropionic Acid – AppNote

Hydrophilic acid retention on the Cogent Diamond Hydride™  Dimethylolpropionic Acid is a unique diol molecule with both hydroxyl and carboxyl functionality and presents several challenges in chromatographic analysis. Traditional reversed phase mode may not be viable due to the high hydrophilicity of this organic acid. Also, by lacking chromophores it can be difficult to detect […]

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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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DMAP, 4-Dimethylaminopyridine Analyzed with HPLC – AppNote

Analyzing the Catalyst, DMAP, 4-Dimethylaminopyridine with HPLC This AppNote shows a rapid, reliable, Isocratic Reversed Phase, HPLC Method that was developed for the Analysis of an important Catalyst for many applications and industries: 4-Dimethylaminopyridine (DMAP). Peak: DMAP Method Conditions Column: Cogent Diamond Hydride™, 4μm, 100Å Catalog No.: 70000-7.5P Dimensions: 4.6 x 75mm Mobile Phase: —90:10 Solvent A / […]

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