Leucine & iso Leucine Separated with LCMS – AppNote

Leucine and Isoleucine, Isobaric Compounds Separated for LCMS Detection This Application Note presents a Method for the Analysis of two Branch Chain Amino Acids (BCAA) with nearly identical m/z values. Addition of weak Acetic Acid to the Mobile Phase allows the small difference in the pKas of the two Isobaric Compounds to achieve Resolution. A […]

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Lidocaine in Gel Analyzed with HPLC – AppNote

Assay Method for Solarcaine® Gel Extract This AppNote shows how Lidocaine Hydrochloride (HCL) from a gel extract can be easily analyzed. The compound has a tertiary amine but excellent Peak shape is achieved; which is often difficult to obtain for analytes like this. A Gradient was used to ensure components from the gel extract matrix […]

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Lipophilic Peptides Analyzed with HPLC – AppNote

Simple Gradient for Non-Polar Peptides  In this Method, we demonstrate the Retention of several Hydrophobic Peptides without the use of a typical Reversed Phase Column. Peak: 1. Gly-Tyr ( Glycyl-L-tyrosine ) 2. Val-Tyr-Val ( Valyltyrosylvaline ) 3. Met enkephalin ( Metenkefalin ) 4. Leu enkephalin 5. Angiotensin II Method Conditions: Column: Cogent Diamond Hydride™, 4μm, […]

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Lisinopril Analysis with HPLC – AppNote

Retention and Peak Shape for Highly Polar Compound As a highly hydrophilic compound, Lisinopril is not well-suited to Reversed Phase Methods. The USP assay method for Lisinopril uses a highly Aqueous Mobile Phase (96% 2.76 g / L Monobasic Sodium Phosphate adjusted to pH 5.0 / 4% Acetonitrile) in Reversed Phase with an L7 Column. […]

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LSD and Codeine – AppNote

LCMS Method for Spiked Urine Sample LSD and codeine may be present in Urine samples and methods are needed for their detection in these matrices. The extracted Ion Chromatograms (EICs) for both analytes are shown in the figure and illustrate excellent chromatographic as well as Mass Spectral Separation. The Method in this AppNote produces high […]

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Maleic & Fumaric Acids Analyzed with LCMS – AppNote

Isobaric Organic Acids Separated and Identified Low molecular mass, Isobaric Acids were Retained, Separated and Identified in this simple Method. The Separation of the two compounds with identical mass is necessary in order to determine which of the acids is present in the sample matrix by the Mass Spectrometer. The Method presented was found to […]

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Maleic & Fumaric Isobaric Acids Analyzed with LCMS – AppNote

Separation & Detection of Isobaric Compounds Two Isobaric Acids, Maleic and Fumaric (115 m/z) are separated when using the solvents below and a Gradient Method. Solvent A used in this application note contains 50% Methanol which is recommended when biological samples are used. This Method is reproducible and is fast to equilibrate between gradient runs […]

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Melamine Analyzed with LCMS – AppNote

Detect Trace Levels of Melamine The unique chemical properties of Melamine (base functionalities) present challenges in obtaining Retention and adequate Sensitivity, when using standard HPLC Columns. In this Method, Melamine is Analyzed under Aqueous Normal Phase (ANP) conditions with an increased Sensitivity making it possible for detection at Trace Levels. Peak: Melamine m/z 127 (M+H)+ […]

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Methionine and Glutamic Acid Analyzed with LCMS – AppNote

Non Derivatized Amino Acids From Synthetic Urine Analyzed Amino Acid analysis is generally performed with HPLC along with a time consuming Pre-Column Derivatization process that is very tedious and adds cost. Using this Method, the Derivatization step is not necessary and the compounds are easily quantified. L- Glutamic Acid can be an extremely difficult Amino […]

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Methionine-Enkephalin Analyzed with LCMS – AppNote

Selective Method for an Opiate like Peptide In this AppNote, the developed Method was used to analyze Methionine-Enkephalin (MEK), the resulting Peak shape was Symmetrical. and the Retention time was more than adequate. In addition to the intact peptide, the Gradient method was also designed to analyze its metabolites and enzyme inhibitors. This Gradient Method […]

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Method Transfer for Cyclobenzaprine – AppNote

Increased Efficiency: 4 μm to 2.2 μm The two chromatograms compare the retention and efficiency of a Cyclobenzaprine peak using two types of Cogent Diamond Hydride Columns. Figure A uses a near-UHPLC 2.2 µm phase while Figure B uses a standard 4 µm particle size column. The results show consistent retention between the two phases […]

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Methotrexate Analyzed with LCMS- AppNote

Methotrexate an Anti-Neoplastic and Anti-Tumor Drug The powerful anticancer drug, Methotrexate (4-Amino-N10- Methylpteroyl Glutamic Acid) acts as an antimetabolite and is used for the treatment of many neoplastic diseases including acute leukemia, osteosarcoma, non-Hodgkins lymphoma, and breast cancer. There is a great interest in pharmacological studies and clinical monitoring of Methotrexate. A quadrupole Mass Spectrometer […]

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Metoprolol Tartrate Analyzed with HPLC – AppNote

Excellent Peak Shape for an Organic Amine This Assay is easy to perform with a well-retained analyte Peak. Since Peak Tailing can often be an issue with organic amines, such as this compound, using conventional HPLC Columns it is notable that this Method produces a very Symmetrical Peak. In the Chromatogram below, an overlay of […]

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Metronidazole Topical Cream Analyzed with HPLC – AppNote

Retention for Highly Polar API This API has very little Hydrophobic character and is not readily retained by Reversed Phase. The Peak that was obtained had excellent efficiency and symmetry. Three runs are shown in the figure in order to illustrate the repeatability of the data. Peak: Metronidazole Method Conditions Column: Cogent Diamond Hydride™, 4µm, […]

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Milk Extract Analyzed by LCMS – AppNote

Analysis of Composition using LCMS Milk components are polar compounds that are not well retained or resolved by traditional Reversed Phase Chromatography. In addition, many of the compounds do not contain strong chromophores, resulting in low specificity and sensitivity in UV detection. The analysis using the Cogent Diamond Hydride Column and MS detection provides separation […]

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Molnupiravir Analyzed with an Inverse Gradient in HPLC – AppNote

Oral Anti-Viral Medication – Aqueous Normal Phase Method Molnupiravir can be analyzed using this simple “inverse gradient” HPLC Method shown below.  This is offered as an orthogonal approach for a Reversed Phased Method and will show various polar impurities not detected by Reversed Phase HPLC.   Peak: Molnupiravir Method Conditions: Column: Cogent Diamond Hydride™, 4µm, 100Å […]

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Morphine Sulfate Tablet Analyzed with HPLC- AppNote

Excellent Peak Shape for Tertiary Amine Morphine can be a very problematic compound for analysis with HPLC. It has a tertiary amine and has a tendency to tail under some conditions. USP assay methodology uses phosphates, alkylsulfonates, or other LC-MS incompatible agents in the Mobile Phase. This Method uses only Formic Acid and can be […]

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Moxifloxacin Hydrochloride Analyzed with HPLC – AppNote

Simple and Precise Retention of a Polar Antibiotic  Moxifloxacin Hydrochloride requires the addition of buffers as well as ion pair reagents to aid in retention on regular Reversed Phase Columns per the current USP Method. In this simple isocratic Method, we retain this polar compound with good Run-to-Run Precision.  (%RSD = 0.22, SD below 0.004.)  This […]

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Neomycin Sulfate Analyzed with LCMS – AppNote

Neomycin presents a number of challenges to routine Chromatographic Analysis. It lacks Chromophores and therefore is difficult to detect using conventional HPLC techniques and retention in traditional Reversed Phase mode may not be viable due to its high polarity.  However, use of the Cogent Diamond Hydride Column in conjunction with a Mass Spec helps circumvent […]

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Nucleoside Analogs Analyzed with HPLC – AppNote

Polar and Non-Polar Anticancer Prodrugs Analyzed in the Same Run In this Method, we display Retention of both polar and nonpolar compounds in a simple Isocratic run. Adjustment of the aqueous to organic ratio can be used to make either the polar or more hydrophobic compounds elute first. In the example presented, the more hydrophobic […]

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Nucleotides Analyzed with LCMS – AppNote

Internal Only- Archived  UDP-Glucose, UDP-Galactose and Galactose-1-phosphate from Blood Cells For most physiologically relevant Nucleotides, use of Mass Spectrometry is the most practical approach for the analysis with biological samples. With Mass Spec, it is essential to Separate UDP-Glucose and UDP-Galactose chromatographically because they are Isomers or Isobaric Compounds, (same molecular weight) and cannot be […]

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Octreotide Analyzed with LCMS – AppNote

Retained Isocratically or With a Gradient This cyclic Octapeptide can be retained with simple Isocratic Mobile Phase conditions of 50:50 Solvent A / Solvent B (see Figure I). In addition, use of a Gradient produces a sharper Peak if desired (Figure II). Note, that the Gradient starts at an unusually high percent of Water (50%) […]

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Organic Acids Analyzed with LCMS – AppNote

An Important Class of Compounds Organic Acids, an important class of Metabolites, are very well Retained and Separated in this Method. Figure A shows the combined EICs for five small Organic Acids: Maleic, Aconitic, Fumaric, Citric and Oxaloacetic Acids. Two of these acids are isobaric compounds; Maleic and Fumaric Acids (115 m/z) and are separated […]

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Organic Bases and Isomers at a Low pH – AppNote

Separation of Four Amine Containing Test Solutes The four test solutes selected for this application are both well-retained and well-resolved. In particular, the separation between the two isomers is readily accomplished, which may be difficult to achieve in Reversed Phase. This Method uses only Formic Acid as the Mobile Phase additive and is LCMS compatible. […]

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Oxymetazoline HCL Analyzed with HPLC – AppNote

Polar API Separated from a Matrix Peak Oxymetazoline can be difficult to obtain a good Peak Shape with conventional HPLC Methods, and the USP System Suitability requires that the Tailing Factor be not more than 2.0. Also, the USP Method calls for a Cation Exchange (L9) Column for the Assay. This Method produces excellent Peak […]

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pH Gradient to Separate Vitamins – AppNote

Separation of Ascorbic Acid, Riboflavin, Pyridoxine, & Thiamine This LCMS compatible Method shows excellent Separation and Retention for all four analytes. If the analysis were done by Reversed Phase, LCMS incompatible ion pair agents would likely be required to get this type of separation. Ascorbic Acid was found to have better Retention near neutral pH […]

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Pharmaceuticals in Plasma Analyzed with LCMS – Extended AppNote

Acetyl-L-Carnitine, Codeine, Hydroxybupropion, Isotrentinon, Warfarin One of the most important applications of LC-MS today is the quantitative analysis of Plasma Samples. Pharmaceuticals and their metabolites may need to be monitored in Plasma in fields such as clinical testing, Forensics, and Toxicology studies. Advancements in LC-MS capabilities have enabled new possibilities for these types of analyses, […]

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Phenylephrine HCL Tablet Analyzed with HPLC – AppNote

Analysis of Phenylephrine, a Highly Polar Compound Phenylephrine was first studied under various Reversed Phase conditions using a C18 Stationary Phase, but even using a 95% Aqueous Mobile Phase a Retention factor of no more than 0.6 could be obtained. With such low retention, it may be difficult to Separate Phenylephrine from excipients or other […]

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Phenylglycine & Phenylalanine Analyzed with HPLC – AppNote

Analysis using a Simple Mobile Phase. In this Method two important amino acids: L-(+)–alpha-phenylglycine and L-phenylalanine, were Separated. C18 Columns used today which may be present in every Analytical Laboratory may not be able to Retain underivatized Amino Acids. They usually elute at or near the “void volume” with other polar compounds. Peaks: 1. L-(+)-Alpha-Phenylglycine […]

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Phosphorylated Sugars Analyzed by LCMS – AppNote

Sucrose-6-phosphate, Glucose-6-phosphate, & Trehalose-6-phosphate Central Metabolic Intermediates in Plants Sugar Phosphates are very highly polar Metabolites and may be difficult to retain using traditional Reversed Phase Methods. In addition, S6P and T6P are Isobaric Compounds so it is very important to separate these two Phosphorylated Sugars for MS Detection. G6P is easily distinguished by its […]

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Phosphorylated Sugars Analyzed with LCMS – AppNote

Nucleotide Sugars UDP, ADP, TDP and CDP UDP Hexanolamine was used as the Internal Standard in the analysis of Nucleotide Sugars in this rapid analysis Method. The Mobile Phase was designed to maximize the detector response in LCMS for maximum Chromatographic Efficiency. The simple “Inverse Gradient” which produces an Aqueous Normal Phase (ANP) HPLC Method, […]

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Prasugrel Analyzed with HPLC – AppNote

Comparison of Diamond Hydride to C18 HPLC Column Prasugrel has a tertiary Amine and therefore tails in many Reversed Phase methods and conventional HPLC columns. Figure A shows how a sharp and symmetrical peak can be easily obtained with the Cogent Diamond Hydride Column using an MS-compatible Mobile Phase. Figure B shows the peak that […]

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Proline: Trans-4-Hydroxy-L-Proline by LCMS – AppNote

Rapid, High Efficiency Method by LCMS This method is highly specific, efficient and fast for the analysis detection, small volumes of the reaction mixture can be injected and of trans-4-hydroxy-L-proline. Due to the high specificity of the the amount of the produced compound can be determined. No derivatization is required for the detection of this […]

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Propantheline Bromide HPLC Methods- AppNote

Orthogonal Methods without Ion-Pairing Agents Click HERE for Column Ordering Information. In the USP assay method for Propantheline Bromide tablets, Sodium Dodecyl Sulfate (SDS) is used as an Ion-Pairing Agent in the Mobile Phase. The long alkyl chain of this additive makes it particularly difficult and time consuming to load and remove from the HPLC […]

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Propranolol in Urine Analyzed by LCMS – AppNote

Excellent peak shape for basic compound The developed method permitted analysis of Propranolol in urine after simple sample preparation. The analysis is performed at a high concentration of Acetonitrile (Acetone may be used as well). The peak shape obtained for this basic compound was excellent. The method could be easily applied to analysis of Propranolol […]

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Purine Nucleotides Analyzed with HPLC- AppNote

Separation of Xanthosine -5’-Monophopate and Guanosine -5’-Triphosphate This Method achieves the separation of Nucleotides XMP from GTP. The analysis of these polar compounds is achieved at high concentration of an organic Solvent as part of the Mobile Phase which provides increased Sensitivity. Retention times were very Reproducible with %RSD approximately 0.4, even when Red Blood […]

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Pyrilamine & 4-Amino-3-Chloropyridine – AppNote

Unique Selectivity on an Amide Stationary Phase The Cogent Amide Column offers unique selectivity that may not be readily attainable with other phases. Two test solutes shown in this application note (Pyrilamine and 4-Amino-3-Chloropyridine) were baseline separated on the Cogent Amide Column (Figure A), but  they co-eluted with no resolution on a different Cogent Column […]

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Pyrilamine Maleate Analyzed with HPLC – AppNote

Separation of Pyrilamine with a Simple Isocratic Method Pyrilamine is a challenging compound to analyze with HPLC because it contains several amine groups that can contribute to peak tailing. In this Method, an excellent Peak shape is obtained for Pyrilamine with a simple isocratic Mobile Phase. Data is shown for two lots of Columns in […]

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Ranitidine Tablet Analyzed with HPLC – AppNote

Excellent Peak Shape of API From an OTC Tablet Ranitidine has several amine functional groups that can contribute to significant tailing with Reversed Phase Methods. Data shown below uses an extract from a tablet formulation, illustrating how good Peak shape can be obtained. In addition, three Column lots were used to demonstrate the robustness and […]

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Red Bull Energy Drink Analysis – AppNote

Separation of Taurine and Caffeine with LC-MS A method combining Solid Phase extraction with high performance liquid chromatography–electrospray ionization tandem Mass Spectrometry was developed for the highly sensitive and accurate screening of Caffeine and Taurine in energy drinks. The matrix pretreatment by SPE sample extraction was essential in the analysis of Taurine. The matrix did […]

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Remdesivir and GS-441524 a ProDrug – AppNote

Retention of polar and non-polar drug compounds in ANP Remdesivir, an antiviral drug, and GS-441524, a pro-drug converted in the body as a ribonucleotide analog, is as a mixture that may be difficult to analyze in routine chromatographic analysis. As GS-441524 is polar, is makes it more difficult to retain in typical Reversed Phase conditions. […]

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Retain Highly Polar Compounds with LCMS – AppNote

Analysis of Amino-Caproic Acid by a Simple LCMS Method 6-Amino-N-Caproic Acid is an active pharmaceutical ingredient used in massive thrombolysis and proteolysis secondary to metastatic carcinoma of the prostate. It is also a potent in vitro inhibitor of fibrinolysis. After oral administration 6-Amino-N-Caproic Acid enhances the uptake of labeled Fibrinogen in both the Walker and […]

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Ribose and Xylose – AppNote

Sugars can be difficult to analyze with HPLC due to their polarity. Columns with amine ligands are often used for retention of simple sugars like Ribose and Xylose, but they have a number of drawbacks. The amine group can form Schiff bases with Aldehydes in the sample, resulting in irreversible deactivation of the ligand’s retention […]

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Sarcosine Analyzed with LCMS – AppNote

Separation of Potential Urine Biomarker from Isobaric ß-Alanine This developed LCMS method can separate Sarcosine from Beta- Alanine in serum and urine samples without using labor intensive sample derivatization. Since Sarcosine is considered a potential biomarker for prostate cancer risk and aggressiveness, it is essential to resolve and accurately quantify this compound in the presence […]

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Separation of Adenine Nucleotides – AppNote

Separation of AMP, ADP, ATP with HPLC The figure shows a separation of three energy nucleotides. All three are baseline separated in order of increasing polarity as is expected when ANP chromatography is used. It is worth noting that the retention of nucleotides increased as the buffer concentration was increased (data not shown). 16.0mM concentration […]

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Separation of Inosine Nucleotides – AppNote

IMP, IDP, and ITP Analyzed with HPLC The figure shows the optimized separation of ITP (Inosine 5’-monophosphate), IDP (Inosine 5’-diphosphate) and IMP (Inosine 5’-triphosphate) in the order of increasing Phosphate content similar to anion exchange. The presence of at least one impurity near ITP and possibly a second near IMP precluded accurate determination of peak […]

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Separation of Nucleotides – AppNote

AMP, NAD, NADP, and GTP on UDA Column Nucleotides are not well retained under Reversed Phase conditions due to their highly polar nature (the presence of one or more Phosphate groups). In this method, weak cation-exchange interactions can provide additional retention/selectivity along with the ANP retention of the Hydride surface. Peaks: 1. AMP – Adenosine […]

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Separation of Polar Solutes with HPLC – AppNote

Niacin, Riboflavin, Folic Acid, Pyridoxine, Metformin, Thiamine The Cogent Diol Column is a good addition to the TYPE-C™ Silica line of HPLC stationary phases. Here, a variety of common polar analytes are well-retained and separated. Peaks: 1. Ascorbic acid 2. Niacin 3. Riboflavin 4. Folic acid 5. Pyridoxine 6. Metformin 7. Thiamine Method Conditions Column: […]

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Serotonin, Metabolites & Analogs Analyzed with HPLC with LCMS – AppNote

Simple HPLC Separation Method without Fluorescent Tags. A Method using Aqueous Normal Phase (ANP), coupled with ESI Detection (LCMS), was developed for the analysis of Neurotransmitters and their Metabolites because previous HPLC based Methods which are used to analyze NT and Metabolites in biological fluids have several drawbacks. Among other problems, they often require a […]

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