Does MicroSolv have a Policy using Materials Free of BSE/TSE?

Yes, as of October 2021, all MicroSolv products are manufactured completely from synthetic or manufactured materials and do not contain any raw materials produced from, or substances derived from animal origin nor do our products come in contact with animal products during storage and transportation therefore all products produced by MicroSolv are free from Transmissible Spongiform Encephalopathy (TSE) and Bovine Spongiform Encephalopathy (BSE) We further declare that our products are Dioxin Free.

For the purposes of this article, TSE is defined as and includes: Bovine Spongiform Encephalopathy (BSE) occurring in cows (mad-cow-disease), the Scrapie Disease in Sheep, as well as the human form: the variant-Creutzfeldt-Jakob-Disease (vCJD).

MicroSolv BSE TSE Free Letter Oct 2021


Have RSA Vials Been Discontinued and did RSA-Pro Change to RSA-Pro X? – FAQ

Concern if RSA™ vials been discontinued and did RSA-Pro™ change to RSA-Pro X™ is answered.

No, RSA™ Vials the non-surface treated, hydrophilic, borosilicate glass vials do not adsorb basic compounds are still available and remain one of the most popular vials in the market today. They and are still an excellent choice for compounds that adsorb to silanols such as basic drugs.

RSA-Pro™ Vials are no longer available and are replaced by an improved technology in RSA-Pro X™ Vials. RSA-Pro vials and RSA-Pro X vials will perform the same basic function but RSA-Pro X does it better, lasts longer even in the presence of water and can be autoclaved and frozen to -80C.

RSA-Pro X™ Vials are indicated for hydrophilic compounds that will stick to glass structures. Proteins, Peptides and other compounds that will stick to bare glass should be used with RSA-Pro X™. You can think of RSA-Pro X™ as a product improvement over RSA-Pro™ and other “silanized” vials and inserts.


What is the Difference Between RSA-Pro and the new RSA-Pro X Surface Treatments? – FAQ

What is the difference between RSA-Pro™ and vs the new RSA-Pro X™ surface treatments?

The biggest difference is that RSA-Pro™ surface treatment incorporated a vapor deposition type process that used ionic bonds with silanes to achieve hydrophobicity of the RSA glass.

RSA-Pro X™ surface treatment uses a very different, proprietary process of covalently bonding hydrophobic materials to the walls of RSA™ vials. The discontinued, RSA-Pro™ treatment was not very hydrolytically stable and could not be autoclaved or frozen.

RSA-Pro X™ will not lose its hydrophobic nature for years, even in the presence of water and they can be autoclaved and frozen and thawed effectively.


Does MicroSolv Have an Industry Classification (NAICS) Number?

CONFIDENTIAL & FOR INTERNAL USE ONLY
August 31 ,2021

Since there is not an NAICS code specific to our industry, MicroSolv uses NAICS code 424990 as our classification. This is as close a classification as exits, as of today.

Please contact our Marketing Department if you have any questions.

 


How Do Our Customers Distinguish Autosampler Vial Sizes? – FAQ

INTERNAL ONLY.

Volume is not the only way customers distinguish Autosampler vials. They may think of a 12x32mm vial as a 2ml vial. We always want to refer to 2ml vials as “12x32mm, 2ml Screw Top Vials” or “12x32mm, 1.8ml Snap Top Vials” etc.

This gives them complete identification of the size and shape of the vial.


LABaler LinkLabel™ BlueTooth Enable

INTERNAL ONLY

Instructions for creating Super and Sub Scripts with the LinkLabel™, Creating Images, Using Symbols

Creating Super Scripts and Sub-Scripts.

Option One

1.  Download the MTC-Bio APP called Label Pro
2.  Click on the orange ” labelled ‘Symbol’ on the ribbon at the top of the screen
3.  Insert the symbol you wish to use, then add the number
4.  Adjust the placement and size of the number manually

Option Two

1)  Download a 3rd party APP called symbolkeyboard-characterpad .
2) Install the symbolkeyboard-characterpad keyboard;

a. Open Settings
b. Click General
c. Click Keyboard
d. Click Keyboards
e. Click Add new keyboard
f. Select “symbol” (if symbol is not an option, restart your phone and try again
g. Once you have selected symbol, you can now view this keyboard by holding the world symbol on the bottom left of your keyboard (PIC 1)
h. For easiest access, add Superscripts and Subscripts to the top of your list inside the Symbol APP (PIC 2 and 3)

PIC 1:

PIC 2:

PIC 3:

Tips for printing symbols, images and pictures

  • Images can only be printed in black OR white
  • Pictures or images will not show up in shades of grey.
  • The ‘Album” icon is used to access your phone camera


Dantrolene Sodium Analyzed with HPLC with UV – AppNote

Dantrolene Sodium has several amine groups that can interact with lone-silanols causing problematic peak tailing when analyzed with conventional HPLC columns. By utilizing a Bidentate C18™ Column with its Silica Hydride™ surface, challenging compounds like Dantrolene can be readily retained with symmetrical peak shape.

Peak:
 Dantrolene Sodium

Method Conditions:
Column: Cogent Bidentate C18™, 4μm, 100Å
Catalog No.: 40018-75P
Dimensions: 4.6 x 75 mm
Mobile Phase: 60% DI Water / 40% Acetonitrile / 0.1% Formic Acid (v/v)
Injection Volume: 2μL
Flow Rate: 1.0ml / min
Detection: 225 nm
Samples: 0.1 mg/mL Dantrolene Sodium in 50:50 Acetonitrile: DI H2O

Note: Essential to muscle contraction are Ryanodine Receptors that regulate the release of Calcium from the Sarcoplasmic Reticulum of muscle cells. Dantrolene Sodium is a postsynaptic muscle relaxant that lessens the “excitation-contraction” coupling response in these cells. It achieves this by inhibiting Calcium binding to Ryanodine Receptor 1 and decreasing intracellular Calcium concentrations.

Attachment

A384 Dantrolene AppNote pdf 0.1 Mb  Download File


How To Help Customers Chose the Proper Centrifuge.

INTERNAL ONLY:

Centrifuges are operated to separate fluids based on density.  Separation is achieved by spinning a vessel containing the material at a speed fast enough to properly separate the heavier materials from the lighter substances.

Some general questions to ask a potential centrifuge client are:

  1. What size and material of tubes will primarily be used on the centrifuge?
  2. What volume will typically be centrifuged?
  3. What is the max speed (*rpm/rcf) required?
  4. Is refrigeration required?
  5.   Do they have a preference for swing-out or fixed angle rotors?

With this information, you can narrow down the right model and rotor(s).

Other issues to cover are the warranty (The warranty for Benchmark™ is 5 years), programmability, adjustable acceleration and deceleration rates, pre-cool functions, and safety features. For example, Hermle™ centrifuges come with all the safety features expected of a quality centrifuge (imbalance detection, safety lid lock, splash proof display, maintenance-free induction drive motor).

One final feature to cover, if applicable to the model, would be the compatibility of the centrifuge with various rotor options and automatic rotor identification.

*RPM is an abbreviation for Revolutions per minute, RCF is the Relative Centrifugal Force exerted on the contents of the rotor.

Attachments:

centrifuges.png  67 Kb  Download File
Centrifuges.xlsx  11.5 Kb  Download File
Hermle Benchmark Warranty Certificate.pdf  0.2 Mb  Download File


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 these issues. The presented data illustrates how the Compound can be both readily retained, with good run-to-run precision, and adequately detected using Mass Spectrometry.

Peak:
Neomycin 615.3196 n/z (M+H)+

Method Conditions:
Column: Cogent Diamond Hydride™, 4µm, 100Å
Catalog No.: 70000-05P-2 
Dimensions: 2.1 x 50 mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid
B: Acetonitrile / 0.1% Formic Acid
Gradient:

Time (minutes) %B
0 90
0.5 90
4 10
5 10
6 90
10 90

Injection Volume: 5µL
Flow Rate: 0.3 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: 0.1 mg/mL Neomycin Sulfate Reference Standard Solution in Solvent A Diluent

Note: Neomycin is aminoglycoside compound that is used as an antibiotic in various types of topical formulations. It is a component of the popular topical cream Neosporin®, used to pre-vent infections. It was discovered by biochemist and microbiologist Selman Waksman and colleagues.

Cogent HPLC Column Logo

Attachment

No 372 Neomyin sulfate.pdf  0.2 Mb   Download File


Glycosidic Analyzed with LCMS – AppNote

Cyanidine-3-Hexoside, Luteolin 7-O-Glucoside & Malvidin Dihexoside
Click HERE for Column Ordering Information.

Observed in Mesquite Flour extracts, these compounds are Glycosidic Flavones and Anthocyanins. The Glycoside Moieties impart polar characteristics to the analytes, which makes them amenable to Retention by the Aqueous Normal Phase (ANP).

In this Application Note, the Cogent Phenyl Hydride Column was used to obtain Chromatographic Separation. The same column can be used in the Reversed Phase mode as well, highlighting its versatility in the analysis of both hydrophilic and hydrophobic compounds.

Peaks:
A. Cyanidine-3-Hexoside (m/z = 450.116 [M + H]+)
B. Luteolin 7-O-Glucoside (m/z = 331.082 [M + H]+)
C. Malvidin Dihexoside (m/z = 449.108 [M + H]+)

Method Conditions:
Column: Cogent Phenyl Hydride™ , 4um, 100A
Catalog No.: 69020-05P-2
Dimensions: 2.1 x 50 mm
Solvents:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

 Time (Minutes) %B
0 90
10 50
12 50
18 20
20 20
21 90

Injection Volume: 1µL
Flow Rate: 0.4mL / minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: Mesquite flour extracted in DI Water and 70:10 Methanol / DI Water. Extracts were centrifuged and the supernatant was collected.

Note: Mesquite Flour may have several advantages over wheat-based Flour for certain types of people. Firstly, it is gluten-free, which may be pertinent to those with celiac disease or gluten sensitivity. For diabetics, its low Glycemic index makes it a good source of Carbohydrates that that can help keep blood sugar levels stable. It is also high in protein and dietary fiber.

Attachment

No 370 Glycosidic Constituents of Mesquite Flour.pdf   0.2 Mb   Download File


Hydrophobic and Hydrophilic Peptides Analyzed with HPLC – AppNote

Simultaneous Separation using a Capillary Column

The studied analytes comprised some very Hydrophobic and Hydrophilic Peptides with diverse isoelectric points (pI) and thus served as useful probes to explore the potential of this App Note in this field of Bioanalysis.

The figure shows how these Peptides of widely disparate physicochemical properties could be separated in a single run. With these capabilities, laboratories can be more efficient and increase throughout by not having to perform separate analyses for both Hydrophobic and Hydrophilic Peptides using different Columns.

Peaks:
1. H-Gly-Arg-Ala-Asp-Ser-Pro-Lys-OH
2. H-Lys-Gln-Ala-Gly-Asp-Val-OH
3. H-Tyr-Ile-Gly-Ser-Arg-OH
4. H-Trp-His-Trp-Leu-Gln-Leu-OH
5. H-Tyr-Gly-Gly-Phe-Leu-OH
6. H-Tyr-Tyr-Tyr-Tyr-Tyr-Tyr-OH

 

Method Conditions:
Column: Cogent Diamond Hydride™ , 4um, 100A R&D Capillary Column
Dimensions: 0.3mm x 150mm
Solvents:
A: DI Water / 0.5% Formic Acid (v/v)
B: Acetonitrile / 0.5% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 90
5 90
10 70
20 60
20.1 30
30 30

Injection Volume: 0.1µL
Flow Rate: 4µL / minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: 10µg / mL of each Peptide in 50% Solvent A / 50% Solvent B.

Notes: The data and discussion presented here are delineated in greater detail in a full research article. Reference: R.I. Boysen, Y. Yang, J. Chowdhury, M.T. Matyska, J.J. Pesek, M.T.W. Hearn, “Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions.” J. Chromatogr. A, 2011, 1218, 8021–8026.

Attachment

No 371 Hydrophobic and Hydrophilic Peptides Analyzed with HPLC pdf  0.3 Mb  Download File


Domoic Acid Separation with LCMS without Derivatization – AppNote

Several analytical methods have been developed for the quantitative determination of Domoic Acid (DA). However, many of the methods require tedious and labor-intensive derivatization of this compound in order to detect it. A new method based on HPLC and MS detection which does not require derivatization has been developed. A simple linear gradient and typical MS compatible mobile phases were used to retain DA, a potent Neurotoxin.

The advantage of using Cogent Columns is that they equilibrate rapidly when a gradient is used. The method can be used by regulatory agencies responsible for monitoring the occurrence of toxins for analysis of Domoic Acid in seafood samples and other laboratories.

Peak:
Domoic Acid 312.1442 m/z (M + H)+

Method Conditions:
Column: Cogent Diamond Hydride™, 4µm, 100Å
Catalog No.: 70000-05P-2
Dimensions: 2.1 x 50 mm
Solvents:
A: 50% Methanol / 50% DI Water / 0.1% Formic Acid
B: 97% Acetonitrile / 3% DI Water / 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 95
10 30
11 30
12 95

Post Time: 5 minutes
Injection Volume: 1µL
Flow Rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: The stock solution was prepared by dissolving 1.0 mg/mL DA in Methanol diluent. The stock was diluted using 50% solvent A and 50% solvent B mixture for the final concentration 0.5 mg/L. Before injection, solution was filtered using a 0.45 micron Nylon Filter (MicroSolv Tech Corp.).

Note: Domoic Acid, a compound known for over 50 years, was found to have neurological effects when ingested by humans. It is the chemical responsible for amnesiac shellfish poisoning (ASP). Upon ingestion by humans, it can cause diarrhea, dizziness, seizures, permanent loss of short-term memory, and even death. In Canada, the European Union, and the USA, the legal limit is 20 µg DA/g in wet tissues of shellfish.

Attachment

No 130 Domoic Acid.pdf 0.2 Mb  Download File


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%) for an Aqueous Normal Phase (ANP) application, due to the highly polar nature of the molecule.

With its two basic Amino Acid residues, the Octapeptide may lead to tailing due to silanolic interactions on some conventional Silica-based HPLC stationary phases, but here the peak shape is very symmetrical.

Peak:
Octreotide

Method Conditions:
Column: Cogent Diamond Hydride™, 4µm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150 mm
Solvents:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minute) %B
0 50
3 20
6 20
7 50

Injection Volume: 1µL
Flow Rate: 0.4mL / minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: Reference standard solution of Octreotide

Notes: Octreotide mimics the naturally occurring hormone Somatostatin. It is used for the treatment of growth hormone producing tumors and other related medical applications.

Attachment

No 367 Octreotide Analyzed with LCMS pdf  0.1 Mb  Download File


Sugars, Sugar Alcohols & Amino Acids Separation with LCMS – AppNote

Glucose, Mannitol, Aspartic Acid, Alanine and Arginine

Metabolomics analysis encompasses a vast array of molecules with disparate hydrophobic/hydrophilic properties. Many  of these analytes are in fact quite polar and problematic to retain by conventional Reversed Phase Chromatography.

The Cogent Diamond Hydride Column is quite well-suited to their separation. This application demonstrates this property through the separation of five notable polar compounds that may be of interest to metabolomics separations.

Peaks:
1. Glucose
2. Mannitol
3. Aspartic Acid
4. Alanine
5. Arginine

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 50 mm
Mobile Phase:
A: 50% DI Water/ 50% Acetonitrile/ 0.1% Formic Acid/ 100µM Sodium Acetate
B: Acetonitrile/ 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 90
1 90
3 80
4 80
5 50
6 50
7 25
10 25
11 90

Post Time: 5 minutes
Injection vol.: 1μL
Flow rate: 0.6 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer

Notes: Sugar Alcohols differ from Sugars in that the Carbonyl group of the former has been reduced to an Alcohol group. They don’t raise blood sugar levels to the same extent that regular Sugars do and hence are often used in diabetic foods when a sweet taste is still desired. Amino Acids have many biological functions and are the building blocks of Peptides and Proteins.

Attachment

No 366 Sugars Sugar Alcohols and Amino Acids AppNote  0.2 Mb  Download File


Tramadol Separation with Enhanced LCMS – AppNote

LC-MS Compatible HPLC Method for Tramadol 

Tramadol can be retained by Reversed Phase HPLC  but some reported methods require Phosphate buffer additives in the Mobile Phase, which means incompatibility with LC-MS. In contrast, this Method uses Formic Acid as the Mobile Phase additive.

An advantage this method has is that it produces enhanced signal-to-noise compared to Reversed Phase thereby producing better results. High Water content in the Reversed Phase Mobile Phase is often required for retention. Since Water is less volatile than Acetonitrile, it is more difficult to remove in LC-MS, hence also leading to lower sensitivity than this Method.

 

PEAK:
Tramadol, m/z 264.2 (M+H)+

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-10P-2
Dimensions: 2.1 x 100mm
Mobile Phase:
A: DI Water/ 0.1% Formic Acid
B: Acetonitrile/ 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 90
5 90
7 30
8 30
10 90

Injection vol.: 1μL
Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Sample Preparation: Tramadol HCl (50 mg) pill was ground with mortar and pestle into fine powder and was used to prepare 1 mg/mL stock solution in Milli-Q DI Water diluent. Then, the sample was sonicated for approximately 5 minutes prior to 0.45 um syringe filtration.

Note: Tramadol is an analgesic used to treat moderate to moderately severe pain. It can be used in both human and veterinary applications. It is sold under various formulation types and brand names, including Ryzolt®, Ultracet®, and Ultram®.

Attachment

No 363 Tramadol.pdf  0.2 Mb  Download File


Fructose and Glucose in Coca-Cola Analyzed with HPLC & RI – AppNote

Separation of Polar Sugars in Coca Cola©

The high sugar content of soda beverages can be observed in this Chromatographic separation of Fructose and Glucose in cola. The two Sugars are retained and separated with the use of the Cogent Amide Column. Data from reference standards is also shown for peak identity confirmation.

A preconditioning protocol with a TEA-based Mobile Phase was found to improve the peak shape of Fructose. Refractive Index is an ideal choice for this application because the Sugars are UV-transparent and their concentrations are high enough (even with a 5X dilution) to be easily observed with a relatively insensitive detector such as this.

The large Peak at the Solvent front is characteristic of RI detection when the sample diluent differs from the Mobile Phase. In this case, a diluent with higher water content than the Mobile Phase was chosen for the purpose of analyte solubility.

PEAKS:
1. D-Fructose
2. D-Glucose

Method Conditions
Column: Cogent Amide™, 4μm, 100Å
Catalog No.: 40036-05P
Dimensions: 4.6 x 50mm
Mobile Phase:
5% DI Water / 95% Acetonitrile (v/v),
15 min preconditioning with 10% DI Water / 90% Acetonitrile / 0.1% Triethylamine (TEA) (v/v)
Injection vol.: 1μL
Flow rate: 1.0 mL/minute
Detection: Refractive Index
Sample: Coca-Cola soda sample was filtered (0.45µm, nylon) and diluted 1:5 with a Diluent of 50/50 Acetonitrile / DI Water.
t0: 0.8 minutes

Note: Coca-Cola was invented by John Pemberton, who was wounded in the Civil War and consequently became addicted to morphine. He devised a recipe called “Pemberton’s French Wine Coca” that was intended as a medicinal agent and substitute for morphine. When laws were enacted that forced Pemberton to create a non-alcoholic version of the drink, the first formulation of Coca-Cola was born.

Attachment

No 358 Fructose and Glucose in Coca-Cola.pdf 0.5 Mb  Download File


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 was obtained using a Reversed Phase gradient (30–60%B over 5 minutes) using the same Mobile Phase solvents and a Type B silica based C18 Column. Here the effects of residual silanols on the Type B silica based column lead to peak tailing.

Peak:
Prasugrel

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 97
1 97
5 60
6 97

Post Time: 2 minutes
Flow rate: 1.0 mL/minute
Detection: 254 nm
Injection vol.: 1μL
Sample Preparation: 0.1 mg/mL Prasugrel in Methanol diluent
t0: 0.9 minutes

Note: Prasugrel is a platelet inhibitor which was approved by the US Food and Drug Administration in 2009 for the reduction of thrombotic cardiovascular events. It is marketed as Effient®.

Attachment

No 187 Prasugrel.pdf 0.7 Mb Download File


Histamine Analysis in Tuna by LCMS – AppNote

LC-MS method without derivatization

A small amount of Histamine was found in a Tuna sample (Figure A) after an extraction procedure and analysis using the Cogent Diamond Hydride Column and MS detection. In Figure B, a Tuna sample was spiked before the extraction procedure at a level of 0.5 mg/L and Figure C shows the Histamine peak in a spiked extract from the Tuna sample at a level of 1.0 mg/L.

The figures show that the identification of Histamine by mass or retention time is not affected by the Tuna matrix or extracted material. The Histamine content in the Tuna sample was determined based on the calibration curve and it was determined to be 320 ± 4 ng/grams of Tuna (with a %RSD of 0.2 for n=5). The developed protocol after validation can be used for the analysis of this polar compound in a variety of food matrices.

Peak:
Histamine 112.0869 m/z

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: 50% DI Water / 50% 2-Propanol / 0.1% Formic Acid
B: Acetonitrile / 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 80
5 10
7 10
8 80

Post Time: 3 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POSG – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation:
Canned Tuna was purchased from a local supermarket. Three Tuna samples were prepared. The unspiked SPE sample was prepared by homogenizing 5 g of Tuna and 50 mL of DI Water / 0.1% FA in a Waring blender for 10 min at 13,500 rpm. The mixture was then centrifuged at 4000 g for 20 minutes. The supernatant was refrigerated (20 °C) for 10 min, treated by adding dropwise 3 M Ammonia to a pH of 11.0, then centrifuged at 1000 g for 5 min. The resulting supernatant was purified by solid phase extraction (SPE) on a conditioned C18 sorbent and eluted with 2 mL of methanol.

After removal of the Methanol by Nitrogen gas, the extracted sample was re-dissolved in 2.0 mL of DI Water / 0.1 % FA for direct analyses. The spiked SPE samples were prepared by homogenizing 5.0 g of Tuna, 50 mL of DI Water / 0.1% FA, and appropriate amount of 1 mg/mL Histamine stock solution in a Waring blender for 10 minutes at 13,500 rpm. Afterwards, the sample preparation was completed by following procedures for the unspiked SPE samples (i.e. centrifuge, SPE, etc.).

t0: 0.9 minutes

Attachment

No 316 Histamine in Tuna.pdf 0.3 Mb  Download File


Tizanidine HCl Tablet with Precision – AppNote

Excellent Run to Run Precision

Tizanidine has several amine groups which can present problems to chromatographers. Residual silanols on the surface of ordinary Silica-based Columns can electrostatically interact with these groups, causing peak tailing.

The Cogent Amide Column, used in the Application Note is virtually free of residual silanols and therefore a symmetrical peak shape can be obtained. An overlay of five consecutive runs demonstrates the run- to-run precision when using these columns.

Peak:
Tizanidine

Method Conditions
Column: Cogent Amide™, 4μm, 100Å
Catalog No.: 40036-05P
Dimensions: 4.6 x 50mm
Mobile Phase:
A: 90% DI Water / 10% Acetonitrile / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 90
2 90
6 40
7 90

Flow rate: 1.0 mL/minute
Detection: UV @ 230nm
Injection vol.: 3μL
Sample Preparation:
4 mg strength Tizanidine HCl tablet was ground and weighed in a 10 mL volumetric flask. A portion of 50/50 solvent A/ solvent B diluent was added and the flask was sonicated 10 minutes. It was then diluted to mark and filtered with a 0.45µm Nylon syringe filter (MicroSolv Tech Corp). An aliquot was then diluted 1:10 with a diluent of 10% solvent A / 90%.

Note: Tizanidine is a centrally acting α2- adrenergic agonist used to treat spasms, cramping, tightness of muscles, and related conditions. It is available under the trade name Zanaflex® as well as generic versions.

Attachment

No 356 Tizanidine Amide.pdf 0.5 Mb  Download File


Galactose and Sucrose – AppNote

Retention and Separation of Mono and Disaccharides

This application demonstrates the ability of the Cogent Amide Column to retain two highly polar test solutes. Sucrose, for example, has a log P of -3.7 and hence would be unlikely to retain in Reversed Phase. Excellent separation is obtained here for these Mono and Disaccharides. The noisy baseline is simply due to the inherent limitations of the Refractive Index Detector, used in this case due to the lack of chromophores in these molecules. The large peak at 1 minute is from the solvent front.

Peaks:
1. D-Galactose
2. Sucrose

Method Conditions
Column: Cogent Amide™, 4μm, 100Å
Catalog No.: 40036-05P
Dimensions: 4.6 x 50mm
Mobile Phase:
A: 90% DI Water / 10% Acetonitrile / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Flow rate: 1.0 mL/minute
Detection: Refractive Index
Injection vol.: 4μL
Sample Preparation: D-Galactose and Sucrose reference standards (1 mg/mL) in diluent of 30% Acetonitrile / 70% DI Water (v/v)

Note: Galactose is a monosaccharide and Sucrose (common name “table sugar”) is a disaccharide. Although a ubiquitous component of sweet foods and beverages today, refined Sucrose was once considered a luxury in many parts of the world.

Attachment

No 355 Galactose and Sucrose pdf 0.4 Mb  Download File


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 LC-MS if needed. With more complex fluoxetine analyses such as those of plasma extracts, LC-MS may be required.

Lot to lot reproducibility of the Cogent Amide Column is highly consistent and reliable. The chromatogram overlay shows results from three different synthesis batches, with a retention time %RSD of 1.2%.

Peak:
Fluoxetine

Method Conditions
Column: Cogent Amide™, 4μm, 100Å
Catalog No.: 40036-05P
Dimensions: 4.6 x 50mm
Mobile Phase:
A: 90% DI Water/ 10% Acetonitrile / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 93
2 93
6 60
7 93

Flow rate: 0.8 mL/minute
Detection: UV 228nm
Injection vol.: 0.5μL
Sample Preparation:
20 mg strength Fluoxetine capsule contents were added to a 50 mL volumetric flask with a portion of 50:50 solvent A:B diluent, sonicated 10 minutes, and diluted to mark. Then an aliquot was filtered through a 0.45 µm Nylon syringe filter (MicroSolv Technology Corp.) and used for injections.

Note: Fluoxetine is a widely prescribed antidepressant which acts by selective inhibition of presynaptic serotonin reuptake. In addition, Fluoxetine can also act as a noncompetitive antagonist of nicotinic acetylcholine receptors. Sold as a racemic mixture, Fluoxetine’s R and S forms show similar efficacy in vivo, and its binding affinity has been shown to be largely stereo independent.

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Ribose and Xylose – AppNote

Sugars can be difficult to analyze by 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 functionality.

Poor robustness and column life have been reported for Amine columns for this reason. The Cogent Amide Column avoids this problem because its ligand is less chemically reactive than an amine, while still obtaining good retention and separation of the two sugar analytes.

Peaks:
1. D-Ribose
2. D-Xylose

Method Conditions:
Column: Cogent Amide™, 4 μm, 100 Å
Catalog No.: 40036-10P
Dimensions: 4.6 x 100mm
Mobile Phase: 95% Acetonitrile / 5% DI Water / 0.1% Triethylamine (TEA) (v/v)
Flow Rate: 0.5 mL / minute
Detection: Refractive Index
Injection Volume: 5ul
Sample Preparation: D-Ribose and D-Xylose reference standards (3 mg/mL) in diluent of 50% Acetonitrile / 50% DI Water / 0.1% TEA (v/v)

Note: Ribose and Xylose are aldopentoses that differ only by a chiral center. In addition to the open chain forms, these sugars exist in equilibrium with ring forms (five or six membered) as well as α and β anomers. Both sugars are highly polar and not generally suitable for conventional Reversed Phase retention.

Attachment

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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 using otherwise equivalent method conditions (Cogent Diamond Hydride™, Figure B). The presence of the Amide ligand provides additional selectivity that can make a significant difference in resolving closely- eluting compounds such as these.

Peaks:
1. Pyrilamine
2. 4-Amino-3-Chloropyridine

Method Conditions
Column: Cogent Amide™, 4μm, 100Å
Catalog No.: 40036-05P
Dimensions: 4.6 x 50mm
Mobile Phase:
A: 90% DI Water / 10% Acetonitrile / 0.1% Formic Acid (v/v)
B: B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 90
1 90
7 50
8 90

Post Time: 3 minutes
Flow rate: 1.0 mL/minute
Detection: UV 244 nm
Injection vol.: 2μL
Sample Preparation:
100 mg/L Pyrilamine and 4-Amino-3-Chloropyridine reference standards in diluent of 50/50 solvent A/solvent B. Peak identities confirmed with individual standards.

Note: Amine-containing compounds such as Pyrilamine and 4-Amino-3-Chloropyridine can be difficult to analyze using conventional silica- based stationary phases. These columns have residual silanol groups on the surface that can interact electrostatically with Amines, causing peak tailing. Chromatographers use a number of strategies to avoid these issues, such as use of ion pair agents or endcapping. However, Cogent TYPE-C Silica phases are virtually free of silanols, and therefore good peak shapes can be obtained without these workaround method strategies.

Attachment

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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 LC-MS separation of the two compounds is presented using the Cogent Diamond Hydride Column. The two peaks are baseline separated chromatographically, but further specificity is also obtained by the use of extracted Ion Chromatograms (EICs) for the [M+H]+ ion of each compound. Furthermore, the Mobile Phase does not use ion pair agents and hence can be suitable for methods using ELSD or similar detectors.

Peaks:
1. Amikacin, m/z 586.2930 [M+H]+
2. Tobramycin, m/z 486.2664 [M+H]+

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-10P-2
Dimensions: 2.1 x 100mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid
B: Acetonitrile / 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 85
2 85
10 20
11 20
12 85

Flow rate: 0.4 mL/Minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 0.01 mg/mL Amikacin & Tobramycin reference standard solution mix

Note: Amikacin and Tobramycin are Aminoglycoside antibiotics, effective against multi-resistance Gram-negative bacteria. They act by disrupting bacterial protein synthesis but can have significant side effects, including hearing loss and kidney damage. Amikacin is marketed as Amikin® and Tobramycin as Tobrex®.

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Taurine with HPLC & Refractive Index – AppNote

Retention of Highly Polar Compound

The food and beverage industry may need reliable methods for assay for Taurine in their products. However, its analysis is complicated by the lack of chromophores in its structure. LC-MS can be used instead, but many QC labs may not have this instrumentation or might prefer a more simple detection method for routine assays. Here, simple isocratic methods are shown for retention of a Taurine standard.

The increased retention at higher organic content in the Mobile Phase illustrates the chromatographic behavior of the Cogent Diamond Hydride Column for retention of Polar compounds. As a highly Polar compound, Taurine can be difficult to retain by conventional Reversed Phase methods.

Peak:
Taurine

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Flow rate: 1.0 mL/minute
Detection: Refractive Index
Injection vol.: 4μL
Sample Preparation: 8 mg/mL Taurine reference standard in diluent of 80/20 solvent A / solvent B.
t0: 0.9 minutes

Note: Taurine is added to many popular energy drinks today. It is found naturally in animal tissues and is a major constituent of bile.

Attachment

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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 (about 5% difference).

These method conditions can readily be transferred from the 4um column to the 2.2um; the advantage of the 2.o™ phase is the significantly higher efficiency.

Peak:
Cyclobenzaprine

Method Conditions
Column: Cogent Diamond Hydride™, 2.2μm, 120Å
Catalog No.: 70200-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: 90% DI Water / 10% Acetonitrile / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 90
0.5 90
8 40
9 40

Post Time: 3 minutes
Flow rate: 0.3 mL/minute
Detection: UV @ 230 nm
Injection vol.: 1μL
Sample Preparation:
10mg strength Cyclobenzaprine tablet was ground and added to a 50 mL volumetric flask. A diluent of 80/20/0.1 Acetonitrile/DI Water/Formic Acid was added and the flask was sonicated for 30 minutes. It was then diluted to mark, mixed, and filtered with a 0.45 µm Nylon Syringe Filter (MicroSolv Tech Corp)

Note: Cyclobenzaprine is used to relieve muscle spasms and accompanying acute pain caused by various musculoskeletal maladies. Brand names include Amrix®, Flexeril®, and Fexmid®.

Attachment

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Herbicide and Metabolites by LCMS – AppNote

Glufosinate, N-Acetylglufosinate, and Glufosinate Propanoic Acid

Analysis of these Compounds can be problematic with other methods and poor peak shape may occur. In contrast, the Peaks obtained in this method are very sharp and symmetrical and can be applied to food products containing these types of Compounds.

Peaks:
1. Glufosinate m/z 180.0431 [M-H]–
2. N-Acetylglufosinate m/z 222.00 [M-H]–
3. Glufosinate Propanoic Acid m/z 151.00 [M-H]–

Method Conditions
Column: Cogent Diamond Hydride™, 2.2μm, 120Å
Catalog No.: 70200-05P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 10mM Ammonium Acetate
B: 95% Acetonitrile / 5% DI Water / 10mM Ammonium Acetate (v/v)
Gradient:

Time (minutes) %B
0 90
1 90
1.2 5
5 5
6 90

Post Time: 3 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – NEG – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Glufosinate (1720.64 ppm), N-Acetylglufosinate (639.2 ppm), and Glufosinate Propanoic Acid (1302.5 ppm) stock solutions were diluted 1:100 with 4:1 DI Water: Methanol
t0: 0.6 minutes

Note: Glufosinate is an Herbicide which acts by interference with the Ammonia detoxification metabolic pathway. Trade names of formulations featuring the compound include Rely®, Finale®, and Ignite®.

Attachment

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Histamine in Cheese Analyzed by LCMS – AppNote

No Derivatization Required for this Analysis of Histamine

A small but measurable amount of Histamine was found in a Cheese sample (Figure A) after an extraction procedure and analysis using the Cogent Diamond Hydride Column with MS detection. Two spiked Cheese samples were also analyzed. In Figure B, the Cheese sample was spiked before the extraction procedure at a level of 0.5 mg/L and Figure C shows the Histamine peak in a spiked extract from the Cheese sample at a level of 8.0 mg/L.

From the figures, it is obvious that the identification of Histamine by mass or retention time is not affected by the Cheese matrix or the extracted material. The Histamine content in the Cheese sample was determined based on a calibration curve and it was calculated to be 500 ± 5 ng/grams of Cheese (with a %RSD of 0.2 for n=5).

Peak:
Histamine 112.0869 m/z [M+H]+

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 50% 2-Propanol / 0.1% Formic Acid
B: Acetonitrile / 0.1% Formic Acid
Gradient:

Time (Minutes) %B
0 80
5 10
7 10
8 80

Post Time: 5 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Grated Parmesan Cheese was purchased from a local supermarket. Three Cheese samples were prepared. The unspiked SPE sample was prepared by homogenizing 5g Cheese and 50 mL DI Water / 0.1% Formic Acid in a Waring blender for 10 minutes at 13,500 rpm. The mixture was then centrifuged at 4000 g for 20 minutes. The supernatant was refrigerated (20°C) for 10 min, treated by adding dropwise 3 M ammonia to a pH of 11.0, and then centrifuged at 1000 g for 5 min. The resulting supernatant was purified by Solid Phase Extraction (SPE) on a conditioned C18 sorbent and eluted with 2 mL of Methanol. After removal of the Methanol by Nitrogen gas, the extracted sample was re-dissolved in 2.0 mL of DI Water / 0.1 % Formic Acid for direct analyses. The spiked SPE samples were prepared by homogenizing 5.0 g of Cheese, 50 mL of DI Water / 0.1% Formic Acid, and an appropriate amount of 1 mg/mL Histamine stock solution in a Waring blender for 10 min at 13,500 rpm. Afterwards, the sample preparation was completed by following procedures for the unspiked SPE samples (i.e. centrifuge, SPE, etc.).
t0: 0.9 minutes

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Warfarin, Hydroxybupropion, and Codeine by LCMS – AppNote

Analysis in Spiked Blood Samples

In this application, three pharmaceuticals/metabolites are separated using the Cogent Diol 2.o Column. The blood sample illustrates the suitability of the Column for analysis of more complex matrices.

Use of Acetone in the Mobile Phase was found to be appropriate for this LC-MS method. As a less expensive and less toxic alternative to Acetonitrile, Acetone presents several advantages to the chromatographer.

Peaks:
1. Warfarin, m/z 309.1121 [M + H]+
2. Hydroxybupropion, m/z 256.1099 [M + H]+
3. Codeine, m/z 300.1594 [M + H]+

Method Conditions
Column: Cogent Diol 2.o™, 2.2μm, 120Å
Catalog No.: 40260-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetone / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 90
3 30
5 30
6 90

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Stock solutions of each analyte were prepared at 1 mg / mL concentrations using a Methanol diluent. Working solutions were then prepared from the stock solutions at concentrations of 1 micro g / mL. All solutions were stored at -20°C. Solutions used for spiking were prepared at 0.500 micro g / mL concentrations. For Blood samples, 0.2 mL Blood in a 2 mL plastic tube was mixed with 0.2 mL Methanol and 0.2 mL spiking solution. The samples were vortexed for 1 minute and centrifuged for 10 minutes at 13,000 rpm. The final solutions were prepared by diluting 0.2 mL supernatant with 0.5 mL Water / 0.1% Formic Acid.

Note: Warfarin is used in the prevention of Thrombosis. Hydroxybupropion is the active metabolite of Bupropion, a smoking cessation aid. Codeine is an opiate compound that is a common ingredient in many Cough Syrup formulations.

Attachment

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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 Efficiency for both Peaks.

Peaks:
1. LSD 324.207 m/z [M + H]+
2. Codeine 300.1594 m/z [M + H]+

Method Conditions
Column: Cogent Diamond Hydride 2.o™, 2.2μm, 120Å
Catalog No.: 70200-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetone / 0.1% Formic Acid (v/v)
Gradient:

Time (Minutes) %B
0 85
3 30
5 30
6 85

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Urine spiked with 5ppm LSD and Codeine standard solutions

Note: Lysergic Acid Diethylamide (LSD) was synthesized by Swiss chemist Albert Hofmann while researching Ergot Alkaloid Derivatives. It is highly potent and produces significant psychedelic effects when ingested. It is currently a Schedule I drug in the U.S. Codeine is an opiate used for analgesic, antitussive, and other effects. It may be obtained from over-the-counter formulations like Cough Syrup and therefore has potential for abuse.

Attachment

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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 the Diamond Hydride 2.o Column, which is well-suited to polar compounds such as these. A spiked Urine sample was used to demonstrate the applicability of the data to clinical studies.

Peaks:
1. Triamterene 254.1149 m/z [M + H]+
2. Amiloride 230.0552 m/z [M + H]+

Method Conditions
Column: Cogent Diamond Hydride 2.o™, 2.2μm, 120Å
Catalog No.: 70200-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 80
3 30
5 30
6 80

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Urine was spiked with Amiloride and Triamterene standard solutions and filtered with 0.45um Nylon Syringe Filter (MicroSolv Tech Corp.).

Note: Amiloride and Triamterene are Potassium-sparing Diuretics. Trade names include Dyrenium® and Midamor®. They are also sometimes combined with Hydrochlorothiazide, a Diuretic drug.

Attachment

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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- Phosphate forms). Figure C shows two Inosine compounds, one with an additional Methyl group, being separated.

Peaks:
A: Thioguanine (6-TG) at m/z = 168.0338 [M+H]+  
B: 6-Thioguanosine -5´-Phosphate (TGMP) at m/z =380.3, 6-Thioguanosine -5´-Diphosphate (TGDP) at m/z= 460.3
C: 6-Methyl-Thioinosine-5´-Monophosphate (meTIMP) at m/z = 379.3 and 6-Thioinosine-5´-Triphosphate (TITP) at m/z = 525

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 50% Methanol / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 100
12 30
14 30
15 0
19 0
20 100

Post Time: 2 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 0.4 mg/mL solutions in DI Water. For MS analysis, samples were diluted 1:100 into 50% Acetonitrile / 50% DI Water mixture. Before injection, samples were filtered through a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).

Attachment

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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 samples.

The method was found to be linear from 0.1 – 10 ng/mg (r2 > 0.999). Recovery of Benzylpiperazine was found to be greater than 95%. Matrix effects were determined to be < 6%.

The concentration of Benzylpiperazine in spiked samples of hair was determined in range from 1.2 – 1.5 ng/mg. The procedure after validation will be useful for laboratories performing routine analysis of drugs of abuse.

Peaks:
A: Benzylpiperazine 177.1386 m/z [M+H]+
B: 5 injections of the sample

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 80
3 30
6 30
10 80

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Extraction (SPE) was performed on cartridge I (Clean Screen Xcel™ purchased from UCT Bristol, PA, USA), preconditioned with 3mL of Methanol, 3 mL of DI Water, and 1 mL of pH 6 buffer prior to sample loading. 10 mg samples of hair (controls, and spiked test samples) were digested in 1 mL of 1 M Sodium Hydroxide for 1 hour at 70˚C. The samples were cooled, and 100 µL of Acetic Acid (Glacial) was added.

Each solution was adjusted to pH 6 with 0.1 mM Ammonia and applied to the SPE column. After loading the samples, each sorbent was washed with DI Water, Acetic Acid (0.1 M), and Methanol (3 mL of each, respectively). Each SPE Column was dried and eluted with 3 mL of Methylene Chloride / Isopropanol / Ammonium Hydroxide (78:20:2). After elution, solvents were evaporated and 200 µL of Mobile Phase was added. The samples were used for analysis by LCMS.

Attachment

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Tizanidine HCl HPLC Method Transferred to Near UHPLC – AppNote

Use of Near UHPLC Column for Improved Results

This challenging compound has several amine groups and can be problematic to analyze by HPLC. Here a good peak shape is obtained using the Cogent Diamond Hydride 2.o Column. The
efficiency is higher on the 2.2µm phase compared to a standard 4µm Column, leading to increased sensitivity. The method conditions are compatible with LCMS as well.

Peak:
Tizanidine HCI

Method Conditions
Columns:
Fig. A: Cogent Diamond Hydride 2.o™, 120Å
Fig. B: Cogent Diamond Hydride™, 4µm, 100Å
Catalog Nos.:
Fig. A: 70200-05P-2;
Fig. B: 70000-05P-2
Dimensions:
Fig. A: 2.1 x 50 mm
Fig. B: 2.1 x 50 mm
Mobile Phase:
—  A: DI Water / 0.1% Formic Acid (v/v)
—  B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient: 

Time (minutes) %B
0 95
1 95
6 40
7 95

Post time: 3 minutes
Injection vol.: 0.2 µL
Flow rate: 0.3mL/minutes
Detection: UV @ 230 nm
Sample: 4mg strength Tizanidine HCl tablet was ground and weighed in a 10mL Volumetric Flask. A portion of 50/50 Solvent A / Solvent B diluent was added and the flask was sonicated 10 minutes. It was then diluted to mark and filtered with a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).
t0: 0.6 minutes

Note: Tizanidine is a centrally acting A2-Adren- Ergic agonist used to treat spasms, cramping, tightness of muscles, and related conditions. It is available under the trade name Zanaflex® as well as generic versions.

Attachment

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Alprazolam (Xanax®) HPLC Method Transferred – AppNote

Benefit of 2.2um Column Analyzing Alprazolam by 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. A) and is further sharpened with the use of a gradient. Furthermore, the method conditions are LCMS compatible and could be applied to a variety of applications involving Alprazolam analysis such as clinical assays of Plasma extracts.

Peak:
Alprazolam (API)

Method Conditions
Columns:
Fig. A: Cogent Diamond Hydride 2.o™, 2.2µm, 120Å
Fig. B: Cogent Diamond Hydride™, 4µm, 100Å
Catalog Nos.:
Fig. A: 70200-05P-2;
Fig. B: 70000-05P-2
Dimensions:
Fig. A: 2.1 x 50 mm
Fig. B: 4.6 x 50 mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient: 

Time (minutes) %B
0 95
1 95
6 50
7 95

Post time: 3 minutes
Injection vol.: 0.2 µL
Flow rate: 0.29 mL / minutes
Detection: UV @ 254 nm
Sample: 15 mg strength Morphine Sulfate tablet was ground and weighed in a 25 mL Volumetric Flask. A portion of 50 / 50 solvent A / solvent B diluent was added and the flask was sonicated for 10 minutes. It was then diluted to mark and filtered with a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).
t0: 0.6 minutes

Attachment

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Vancomycin HPLC Method Transferred – AppNote

Benefits of using 2.2 µm Column for Vancomycin Analysis

The highly polar antibiotic Vancomycin can be readily analyzed with either the standard 4µm Cogent Diamond Hydride or the Cogent Diamond Hydride 2.o Column. Notably higher efficiency is obtained on the smaller particle size Column.

This Method is easy to perform and is LCMS compatible. Three runs were performed on each column in order to demonstrate consistency and show the benefits of using the smaller particle Column.

Peak:
Vancomycin

Method Conditions
Columns:
Fig. A: Cogent Diamond Hydride 2.o™, 2.2µm, 120Å
Fig. B: Cogent Diamond Hydride™, 4µm, 100Å
Catalog Nos.:
Fig. A: 70200-05P-2;
Fig. B: 70000-05P-3
Dimensions:
Fig. A: 2.1 x 50 mm
Fig. B: 3.0 x 50 mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)

Time (minutes) %B
0 70
0.4 70
7.4 10
8.4 70

Post time: 5 minutes
Injection vol.: 0.2 µL
Flow rate:
Fig. A: 0.29 mL / minutes
Fig. B: 0.50 mL / minutes
Detection: UV @ 210 nm
Sample:
Stock Solution: 1 mg / mL Vancomycin HCl in 50 / 50 solvent A / solvent B diluent. The solution
was filtered through a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.).
Working Solution: Stock solution was diluted 1:100 with 50/50 solvent A/ solvent B mixture.
t0: 0.4 minutes

Note: Vancomycin is a glycosylated non-ribosomal Peptide antibiotic used to treat Colitis. Vancomycin is often used as a drug of last resort when other antibiotics are rendered ineffective due to developed resistance of bacteria. It is a natural product isolated from Amycolatopsis Orientalis.

Attachment

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Human Plasma Extract Analyzed by LCMS – AppNote

 Urea, Lactodifucotetraose, Trimethylamine, L-carnitine & Choline

Using this Method it is possible to analyze Compounds present in a Human Plasma Samples. The mechanism of retention of these polar compounds on the Column used is unknown at this time.

It is possible to Retain and Separate steroids and other Compounds based on the Shape of the Molecules.

Peaks:
1. Urea 61.0396 m/z [M + H]+
2. Lactodifucotetraose 635.2393 m/z [M + H]+
3. Trimethylamine N-oxide 76.0757 m/z [M + H]+
4. L-carnitine 162.1125 m/z [M + H]+
5. Choline 105.1148 m/z [M + H]+

Method Conditions
Column: Cogent UDC Cholesterol™, 4μm, 100Å
Catalog No.: 69069-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 90
1 90
7 20
11 20
12 90

Post Time: 3 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Proprietary Lyophilized Plasma Sample was reconstituted in 60 µL 80% Acetonitrile / 20% DI Water mixture.

Attachment

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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 mass to charge ratio (m/z).

These three Phosphorylated Sugars were separated using this Method even though the separation is quite challenging, because the structures of two out of three compounds are very similar. Note that a very shallow Gradient is used in this separation.

Peaks:
1. S6P: sucrose-6-phosphate, 421.0753 m/z [M-H]–
2. G6P: glucose-6-phosphate, 259.0224 m/z [M-H]–
3. T6P: trehalose-6-phosphate, 421.0753 m/z [M-H]–

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 10 mM Ammonium Acetate, pH=6.0
B: 90% Acetonitrile / 10% DI Water / 10 mM Ammonium Acetate, pH=6.0
Gradient:

Time (minutes) %B
0 85
3 85
13 80
15 80
18 75
20 60
21 85

Flow rate: 0.4 mL/minute
Detection: ESI – NEG – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 15 µM G6P, 20 µM S6P, and 20 µM T6P were prepared in 10% DI Water / 10% Methanol / 80% Acetonitrile
t0: 0.9 minutes

Note: Starch produced by Plants is an essential material in the human diet. T6P has a function in promoting biosynthetic processes of Starch in Plants, but the exact mechanism is still unknown. The ability to quantify levels of T6P in Plant tissue is of crucial importance in studies of the regulatory role of T6P in Carbon use. The low quantity of T6P in plant matrices makes its detection and quantification a very challenging analytical problem. The developed method can be used in studies of biosynthetic processes.

Attachment

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Uric acid and Metabolites Analyzed by LCMS – AppNote

LCMS Method for Allantoin, 6-Aminouracil, and Uric Acid (UA)

The presented method for analysis of UA and its highly polar metabolites is simple and does not require Mobile Phase additives or pre-column derivatization. The Peaks are symmetrical, and the MS signal is not diminished, as is the case when Ammonium Formate or Acetate is used as a Mobile Phase additive.

The analyzed metabolites are signature end products for UA degradation in the presence of oxidants and can therefore be used as biomarkers for different disease states. Cogent columns can have very fast equilibration between gradient runs and can be successfully used in studies of pathways in human pathology.

Peaks:
1. Allantoin 159.0513 m/z [M+H]+
2. 6-aminouracil 128.0455 m/z [M+H]+
3. Uric acid 169.0356 m/z [M+H]+

Method Conditions
Column: Cogent Diol™, 4μm, 100Å
Catalog No.: 40060-15P-3
Dimensions: 3.0 x 150mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 95
6 30
7 30
8 95

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Standards of Uric Acid and its main metabolites were prepared in DI Water at concentrations of 20 microg /mL each. The sample for injection (a mixture of the three compounds) was diluted by a factor of 3.
t0: 0.9 minutes

Note: Humans lack the enzyme (Uricase) which converts UA to Allantoin. However, the compound still can be detected in human Urine samples. UA can react with an oxidant in a step reaction and the end product is Allantoin. When UA is degraded it can’t be renewed. The presence of Allantoin in Urine is an indicator of a disease state (oxidative stress, renal failure, diabetes, etc.). The Allantoin/UA ratio is also elevated in Down syndrome and chronic lung disease. 6-Aminouracil is a direct product of reaction of UA with Nitric Oxide, which has a role in Cardiovascular disease.

Attachment

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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 and detection of all the compounds of interest. In this AppNote, The Diamond Hydride Column is able to analyze polar compounds in biological matrices.

 

Peaks:
1. Urea 61.0396 m/z [M+H]+
2. Lactose 343.1235 m/z [M+H]+
3. Galactose 181.0707 m/z [M+H]+
4. Lacto-N-Neotetraose 708.2557 m/z [M+H]+
5. Glutamine 147.0764 m/z [M+H]+
6. Creatine 132.0768 m/z [M+H]+
7. L-Carnitine 162.1125 m/z [M+H]+
8. Unknown 105.1148 m/z [M+H]+

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 90
1 90
7 20
11 20
12 90

Post Time: 3 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – POS – PerkinElmer, Flexar SQ 300 Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Milk extract reconstituted in 65 micoL of 80% Acetonitrile / 20% DI Water
t0: 0.9 minutes

Note: Milk was highly susceptible to microbial growth contamination prior to the advent of pasteurization. Named after the French chemist Louis Pasteur who invented it, pasteurization is a technique that extends the shelf life of milk by killing harmful microbes. In addition to his work in the field of microbiology, Pasteur also made significant contributions to our current understanding of chirality through the isolation of enantiomers of tartaric acid crystals.

Attachment

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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 with MS-compatible mobile phase solvents.

Peaks:
1. Niacinamide
2. Riboflavin
3. Pyridoxine
4. Folic Acid
5. Cyanocobalamin

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water / 10 mM Ammonium Formate
B: 95% Acetonitrile / 5% Solvent A
Gradient:

Time (minutes) %B
0 100
2 100
9 50
10 100

Post Time: 2 minutes
Flow rate: 1.0 mL/minute
Detection: UV @ 266 nm
Injection vol.: 2μL
Sample Preparation: Mix of 0.1 mg/L Niacinamide, 0.01 mg /mL Riboflavin, 0.3 mg / mL Pyridoxine, 0.05 mg / mL Folic Acid, 1.0 mg / mL Cyanocobalamin in 50% Solvent A/ 50% Solvent B diluent. Peak identities were confirmed by individual standards.
t0: 0.9 minutes

Note: The word “Vitamin” was originally spelled “vitamine” when it was first coined by biochemist Casimir Funk. It was derived from the words “vital” and “amine” because it was believed at the time that all Vitamins were chemical Amines. The “e” was dropped from the word when it was discovered that this is not the case.

Attachment

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Uric Acid (UA) Metabolites Analyzed by LCMS – AppNote

Allantoin and 6-Aminouracil

The presented method for analysis of UA metabolites in human Urine is very simple and does not require Mobile Phase additives or pre-Column derivatization. The peaks are symmetrical and the MS signal is not diminished as is the case when Ammonium Formate or Acetate is used as the Mobile Phase additive.

The analyzed metabolites are signature end products for UA degradation in the presence of oxidants and can be used as biomarkers for different disease states.

Peaks:
1. Allantoin 159.0513 m/z [M+H]+
2. 6-Aminouracil 128.0455 m/z [M+H]+

Method Conditions
Column: Cogent Diol™, 4μm, 100Å
Catalog No.: 40060-15P-3
Dimensions: 3.0 x 150mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 95
1 95
6-7 30
8 95

Flow rate: 0.4 mL/minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 200 µL of Urine sample was centrifuged at 14,000 rpm for 10 minutes two times. The supernatant was diluted 1: 2 with Acetonitrile and centrifuged again at 14,000 rpm for 5 minutes. The filtrate was injected into LC-MS.
t0: 0.9 minutes

Note: Humans lack the enzyme (Uricase) which converts UA to Allantoin. However the compound still can be detected in human Urine samples. UA can react with an oxidant in a step reaction and the end product is Allantoin. When UA is degraded, it can’t be renewed. The presence of Allantoin in Urine is an indicator of a disease state (oxidative stress, renal failure, diabetes, etc.). The Allantoin/UA ratio is also elevated in Down syndrome and chronic lung disease. 6-Aminouracil is a direct product of reaction of UA with nitric oxide, which has a role in cardiovascular disease.

Attachment

No 290 Uric Acid Metabolites pdf  0.2 Mb   Download File


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: Cogent Diol™, 4μm, 100Å
Catalog No.: 40060-15P-3
Dimensions: 3.0 x 150mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 95
3 95
10 40
12 40
13 95

Post Time: 5 minutes
Flow rate: 0.7 mL/minute
Detection: UV @ 254 nm
Injection vol.: 1μL
Sample Preparation: Mixture of reference standards in diluent of 50 / 50 Solvent A / Solvent B.
t0: 0.7 minutes

Note: B and C Vitamins are hydrophilic and therefore may be difficult to retain in Reversed Phase methods. Metformin is a highly polar compound used for treatment of type 2 diabetes.

Attachment

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Glutathione (GSH) Analyzed by LCMS – AppNote

LCMS Method without Derivatization

The major benefit of this analysis method for Glutathione which uses the Cogent Diamond Hydride Column and MS detection is that it is performed without derivatization, unlike other LCMS approaches. A GSH signal of MS is stable after repeated analyses. The unique design of the ESI offers efficient supersoft ionization which helps to preserve the structure of the analyte.

This Method ensures low variability of the results. After validation, it could be applied to routine analysis of oxidative stress in clinical samples (blood or plasma).

Peak:
Glutathione 308 m/z

Method Conditions
Column: Cogent Diamond Hydride 2.o™, 2.2μm, 120Å
Catalog No.: 70200-05P-2
Dimensions: 2.1 x 50mm
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)
Gradient:

Time (minutes) %B
0 85
2 30
4 30
5 85

Post Time: 3 minutes
Flow rate: 0.3 mL/minute
Detection: ESI – POS – PerkinElmer Flexar SQ 300 Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: 1 ppm of Glutathione in 50% Acetonitrile / 50% DI Water / 0.1% Formic Acid
t0: 0.7 minutes

Note: Glutathione (GSH, Gamma-L-Glutamyl- L-Cysteinyl-Glycine) is a non-protein Thiol compound which plays an important role in protecting calls against oxidative stress. Lower levels of Glutathione were found in different diseases, like cardiovascular disease, cancers, and aging [3,3]. Under oxidative stress, GSH is converted into its oxidized form Glutathione Disulfide (GSSG). A lower level of GSH is an early indicator of disease risk.

Attachment

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Sorbic and Tartaric Acids in Wine Analyzed by LCMS – AppNote

Minimal Sample Preparation

This method doesn’t require complex preparation or extraction of the wine samples. It allows detection of both Acids in a short gradient run, with rapid equilibration of the column after each run.

Calibration curves were constructed in the ranges 1–300ppm and 100–3000ppm for Sorbic Acid and Tartaric Acid respectively. Correlation coefficients for both Acids were close to 0.997. In  the sample of the Red Wine analyzed, it was determined that the concentration of Sorbic Acid was 8 ppm and Tartaric Acid was 500 ppm.

Peaks:
1. Sorbic Acid 111 m/z
2. Tartaric Acid 149 m/z

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
A: DI Water / 16.5 mM Ammonium Acetate
B: 90% Acetonitrile / 10% DI Water / 16.5 mM Ammonium Acetate (v/v)
Gradient:

Time (minutes) %B
0 95
1 95
4 & 9 30
9.5 95

Post Time: 3 minutes
Flow rate: 0.4 mL/minute
Detection: ESI – NEG – PerkinElmer Flexar SQ 300 Mass Spectrometer
Injection vol.: 1μL
Sample Preparation: Red and white wine samples were filtered using a MicroSolv Tech Corp. filter (0.45µm, Nylon) and diluted using 50 / 50 / 0.1 Methanol / DI Water / Formic Acid.
t0: 0.9 minutes

Note: Sorbic acid (SA) is used worldwide as a preservative for various food and beverages, including wine. It is used to inhibit yeasts and molds. However, in Sorbate treated wines Lactic bacteria have been found to convert SA to 2-Ethoxyhexa-3, 5-Diene, which gives the wine an unpleasant geranium like smell. Tartaric Acid is a by-product in wine production. It is important to determine the amount of Tartaric Acid in the evaporated powder or after purification.

Attachment

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Tropicamide and Homatropine Analyzed by HPLC – AppNote

Separation of Anticholinergic Mydriatic Agents

In this Method, Tropicamide and Homatropine are separated with a simple Gradient. Homatropine is significantly hydrophobic yet still retains well.

Furthermore, the Separation between these two solutes is good, illustrating the Selectivity capabilities of this Method. Data is shown for two lots of Columns in order to demonstrate the robustness and precision of this Method.

Peaks:
1. Homatropine
2. Tropicamide

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water / 0.1% Trifluoroacetic Acid (v/v)
B: Acetonitrile / 0.1% Trifluoroacetic Acid (v/v)
Gradient:

Time (minutes) %B
0 95
1 95
6 50
7 95

Injection vol.: 2μL
Flow rate: 1.0mL / minute
Detection: UV @ 220nm
Sample Preparation: 0.1mg / mL Homatropine and 0.01mg / mL Tropicamide reference standards in diluent of 50:50 Solvent A / Solvent B. Peak identities were confirmed with individual standards.
t0: 0.9 minutes

Note: Homatropine and Tropicamide are mydriatic (pupil-dilating) agents used in ophthalmoscopic examinations. Trade names include Homatropaire® for Homatropine and Mydral® for Tropicamide.

Attachment

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Terbutaline and Salbutamol Analyzed by HPLC – AppNote

Separation of ß2-Adrenergic Receptor Agonists

This Method shows how two structurally similar compounds can be readily separated. This is an important characteristic in many types of HPLC Methods since typical separations often involve impurities, degradants, metabolites, and other groups of compounds that have subtle structural differences. These two compounds for instance were found to coelute using Reversed Phase approaches.

The Method shown here is simple and the Isocratic Mobile Phase is easy to prepare. Baseline resolution is obtained for the two compounds. Furthermore, the Method reproducibility is good, as shown in the figure which uses Columns from two different lots.

Peaks:
1. Terbutaline
2. Salbutamol

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase: 5% DI Water / 95% Acetonitrile / 0.1% Trifluoroacetic Acid (TFA) v/v
Injection vol.: 2μL
Flow rate: 1.0mL / minute
Detection: UV @ 220nm
Sample Preparation: 0.1mg / mL Terbutaline and Salbutamol reference standards in diluent of 50:50 0.1 DI Water / Acetonitrile / Trifluoroacetic Acid (v/v).
t0: 0.9 minutes

Note: Salbutamol and Terbutaline are ß2- Adrenergic receptor agonists used for treatment of asthma and pulmonary diseases. Salbutamol is available under the trade name Ventolin®. Trade names for Terbutaline include Brethine®, Bricanyl®, and Brethaire®.

Attachment

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Ranitidine Tablet Analyzed by 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 precision of this Method.

Peaks:
1. Ranitidine
2. Matrix Component

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-7.5P
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water / 0.1% Trifluoroacetic acid (v/v)
B: Acetonitrile / 0.1% Trifluoroacetic acid (v/v)
Gradient:

Time (minutes) %B
0 95
1 95
6 50
7 95

Post Time: 3 minutes
Injection vol.: 2μL
Flow rate: 1.0mL / minute
Detection: UV @ 313nm
Sample Preparation: 150mg strength Ranitidine HCl tablet was ground and weighed in a 50mL volumetric flask. A portion of 50:50 Solvent A / Solvent B diluent was added and the flask was sonicated 10 minutes. It was then diluted to mark and filtered with a 0.45µm Nylon Syringe Filter (MicroSolv Tech Corp.). The filtrate was diluted 1:100 for injections.
t0: 0.9 minutes

Note: Ranitidine is an acid reducer that is available over-the-counter. It works as a histamine H2-receptor antagonist, in contrast to other acid reducers such as omeprazole which are proton pump inhibitors. It is sold under the trade name Zantac®.

Attachment

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