Applications:
Separation of NyQuil Active Ingredients on Primesep C

Primesep C separates the active ingredients in the over-the-counter cold medicine, NyQuil. Succinate and bromate co-elute early, but acetaminophen, pseudoephedrine, doxylamine, and dextromethorphan are baseline resolved. A combination of cation exchange, complex formation, and hydrophobic interactions separate this mixture with a mobile phase of water, acetonitrile (MeCN, ACN) and triethylamine (TEA) phosphate with UV detection at 205 nm.
Condition
Column |
Primesep C, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
TEAPh |
Flow Rate |
1.0 ml/min |
Detection |
UV, 205 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Pseudoephedrine, Doxylamine, Dextromethorphan |
Acetaminophen (Paracetamol)DextromethorphanDoxylaminePseudoephedrine (PSE)
HPLC Separation of Polar Drugs with MS-compatible Method
Acetaminophen is an over-the-counter pain and fever reducer, and a major component of cold and flue remedies. Phenylephrine is a decongestant. Guaifenesin is a mucolytic agent used to relieve respiratory difficulties. These three compounds of cough medication were separated by mixed-mode chromatography on a Primesep C HPLC column. Retention and order of elution for phenylephrine can be changed by buffer concentration and buffer pH. Method can be used for analysis of cough and cold composition during the production and in QC/QA environment. Method is compatible with LC/MS and can be used to analyze these components in biological fluids. This generic HPLC method is robust and reproducible.
Condition
Column |
Primesep C, 3x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
AmAc |
Flow Rate |
0.5 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Pseudoephedrine, Guaifenesin |
Acetaminophen (Paracetamol)GuaifenesinPhenylephrine
Effect of pH on Retention of Basic Compounds on Primesep C Columns
Method for separation of components of cough medication shows how retention for acetaminophen, phenylephrine and guaifenesin is controlled. Order of elution for compounds can be changed based on composition of the mobile phase. The method is compatible with UV, ELSD and LC/MS and can be used for quantitation of drugs in formulation. Primesep C mixed-mode HPLC column is ideal candidate for analysis of cough compositions. Basic compounds are well retained in mixed-mode chromatography without use of ion-pairing reagents. Several companies validated this approach. The method is reproducible and robust and can be used in both production environments and R&D. This HPLC method can be adopted as general approach for analysis of basic compounds in various mixtures.
Condition
Column |
Primesep C, 3x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
AmAc |
Flow Rate |
0.5 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Pseudoephedrine, Guaifenesin |
Acetaminophen (Paracetamol)GuaifenesinPhenylephrine
HPLC Separation of Acetaminophen, Phenylephrine, and Diphenhydramine

Primesep C separates acetaminophen (paracetamol), phenylephrine, and diphenhydramine-- common over-the-counter cold and flu medication ingredients such as Tylenol products—by either an acetonitrile or phosphoric acid gradient. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN), and phosphoric acid with ultraviolet (UV) detection at 210 nm.
Condition
Column |
Primesep C, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
H3PO4 |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Pseudoephedrine, Diphenhydramine |
Acetaminophen (Paracetamol)Phenylephrine
HPLC Separation of NyQuil Active Ingredients
NyQuil is a cough medication produced by Procter&Gamble. It consists of acetaminophen, pseudoephedrine, doxylamine and dextromethorphan. Most of the analytical HPLC methods include either use of ion-pairing reagent or a gradient elution. Mixed-mode chromatography allows to replace long gradient method with short isocratic method. All four compounds are well separated within 3-5 minutes. Methods can be optimized for analysis of other cough composition containing general cough suppressants and pain killers. Method is robust and being validated at several drug companies.
Condition
Column |
Primesep C, 4.6x50 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
TEAPh |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Pseudoephedrine, Doxylamine, Dextromethorphan |
Acetaminophen (Paracetamol)DextromethorphanDoxylaminePseudoephedrine (PSE)
Separation of Drugs in Mixed-Mode HPLC

Obelisc R column can be successfully used for analysis of hydrophobic and hydrophilic basic and acidic drugs and corresponding counter-ions. This approach can be used for analysis of various cold and cough composition, pain killers, and other prescription and over-the-counter medications. Within pH 3-5, both cation- and anion mechanism are present on the column, along with hydrophobic interaction. Selectivity of this mixed-mode separation is controlled by the amount of acetonitrile, buffer concentration and buffer pH. Method is robust and reproducible, and can be used as a common HPLC method for analysis of drug molecules and compositions.
Condition
Column |
Obelisc R, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
AmFm |
Flow Rate |
1.0 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Maleate, Chlorpheniramine, Dextromethorphan |
Acetaminophen (Paracetamol)Dextromethorphan
HPLC Separation of Drugs in Tylenol Cold and Cough Remedies

Components of Tylenol Cold and Cough Remedy are separated on Obelisc R mixed-mode column. Method can be used to determine compounds in various cough and cold compositions. Neutral (acetaminophen), basic (chlorpheniramine, dextromethorphan and pseudoephedrine) and acidic (maleic acid/Maleate) components are analyzed with perfect peak shape and retention control. Method can be used in production, QC/QA and biological studies for quantitation of various components in pharmaceutical formulations (Advil, Tylenol, Dimetapp, Robitussin, NyQuil, etc)
Condition
Column |
Obelisc R, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
AmFm |
Flow Rate |
1.0 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, Neutral, Basic |
Analyzing Compounds |
Maleate, Chlorpheniramine, Dextromethorphan, Pseudoephedrine, Acetaminophen |
Acetaminophen (Paracetamol)ChlorpheniramineDextromethorphanMaleatePseudoephedrine (PSE)
USP Methods for the Analysis of an Analgesic Mixture Using the Legacy L1 Column
Application Notes: Acetametaphin, aspirin, and caffeine tablets contain not less than 90 percent and not more than 110 percent of the labeled amounts if acetametaphin, asprin, and caffeine according the USP methods. USP HPLC method for separation of acetaminophen, aspirin and caffeine was developed on Legacy L1 column according to US Pharmacopeia methodology. L1 classification is assigned to reversed-phase HPLC column contains C18 ligands. Support for the material is a spherical silica gel with particles size 3-10 um and pore size of 100-120A. Resolution between critical pairs corresponds to rules and specifications of USP.
Application Columns: Legacy L1 C18 HPLC column
Application compounds: Acetaminophen, Aspirin, Caffeine, benzoic acid, and salicylic acid
Mobile phase: MeOH/H2O/AcOH 28/69/3
Detection technique: UV
Reference: USP30: NF35
Condition
Column |
Legacy L1, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeOH/H2O/AcOH 28/69/3 |
Buffer |
AcOH |
Flow Rate |
1.0 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophobic, Ionizable |
Analyzing Compounds |
Acetaminophen, Caffeine, Aspirin, Benzoic acid, Salicylic acid |
Acetaminophen (Paracetamol)AspirinBenzoic AcidCaffeine
Separation of Acetaminophen and 4-Aminophenol on Primesep C
Acetaminophen and 4-Aminophenol ar both aminophenols. Acetominophen or paracetamol is a widely used analgesic and antipyretic. 4-Aminophenol is an intermediate in the synthesis of paracetamol and used in photography as a developer. Both compounds were retained and resolved on Primesep C. Primesep C is a mixed-mode HPLC column which uses reversed-phase and unique complex-forming groups to resolve compounds that might otherwise coelute.
Condition
Column |
Primesep C, 4.6x150 mm, 5 µm, 100A |
Mobile Phase |
MeCN |
Buffer |
TEAPh |
Flow Rate |
1.0 ml/min |
Detection |
UV, 210 nm |
Description
Class of Compounds
|
Drug, Hydrophilic, Ionizable |
Analyzing Compounds |
Acetaminophen, 4-Aminophenol |
4-AminophenolAcetaminophen (Paracetamol)
HPLC Analysis of Paracetamol
Paracetamol (also known as acetaminophen) is a mild analgesic commonly used for relief from headaches and used in many cold and flu medicines. Legacy L1 was used to retain Paracetamol by reverse phase mechanism. Legacy L1 uses embedded C18 groups on porous silica and is useful for many USP HPLC applications. comparisons to Phenomenex columns are available by request.
Condition
Column |
Legacy L1, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeOH - 15% |
Buffer |
AmAc pH 4.0- 42 mM |
Flow Rate |
1.0 ml/min |
Detection |
UV, 257 nm |
Description
Class of Compounds
|
Drug, Hydrophilic, Ionizable |
Analyzing Compounds |
Paracetamol |
Acetaminophen (Paracetamol)
HPLC Separation of Caffeine, Benzoic acid and Acetaminophen
Condition
Column |
Legacy L1, 4.6x250 mm, 5 µm, 100A |
Mobile Phase |
MeOH - 28% |
Buffer |
Acetic Acid - 3% |
Flow Rate |
1.5 ml/min |
Detection |
UV, 275 nm |
Description
Class of Compounds
|
Drug, Acid, Hydrophilic, Hydrophobic, Ionizable |
Analyzing Compounds |
Caffeine, Benzoic acid, Acetaminophen |
&
Acetaminophen (Paracetamol)Benzoic AcidCaffeine
HPLC Method for Analysis of Acetaminophen and Oxycodone

Acetaminophen is a p-aminophenol derivative with analgesic and antipyretic activities. It has weak anti-inflammatory properties, may cause liver, blood cell, and kidney damage. It is a nonprescription medication for mild-to-moderate pain and fever.
Oxycodone is semi-synthetic opioid type drug that is used to help relieve moderate to severe pain. Primesep 200, a reverse phase column, contains embedded acidic ionizable groups and can retain Acetaminophen and Oxycodone. The method is UV compatible and can be used as a general approach for analyzing similar compounds.
Condition
Column |
Primesep 200, 3.2x100 mm, 5 µm, 100A |
Mobile Phase |
Gradient MeCN - 5-50% |
Buffer |
Gradient H3PO4 - 0.05-0.3%, 5 min, 4 min hold |
Flow Rate |
0.5 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Hydrocodone |
Acetaminophen (Paracetamol)Oxycodone
HPLC Method for Analysis of Vicodin (Acetaminophen & Hydrocodone)
Condition
Column |
Primesep 200, 3.2x100 mm, 5 µm, 100A |
Mobile Phase |
Gradient MeCN - 5-50% |
Buffer |
Gradient H3PO4 - 0.05-0.3%, 5 min, 4 min hold |
Flow Rate |
0.5 ml/min |
Detection |
UV, 270 nm |
Description
Class of Compounds
|
Drug, Analgetic, Acid, Hydrophilic, Ionizable, |
Analyzing Compounds |
Acetaminophen, Hydrocodone |
Acetaminophen (Paracetamol)HydrocodoneVicodin
HPLC Separation of Acetaminophen, Caffeine and Pyrilamine maleate

Acetaminophen is a p-aminophenol derivative with analgesic and antipyretic activities. It has weak anti-inflammatory properties, may cause liver, blood cell, and kidney damage. It is a nonprescription medication for mild-to-moderate pain and fever. Caffeine is a Central Nervous System (CNS) stimulant. It is an unregulated and legal drug in most parts of the world. It can be found in the seeds and leaves in a number of plants native in Africa, East Asia and South America. Pyrilamine maleate is a histamine H1 antagonist with hypnotic properties. It has multiple uses such as an anesthetic and is used against allergies. Newcrom R1, a column that takes advantage of the newest technologies, does not contain embedded acidic nor basic ionizable groups and can retain Acetaminophen, Caffeine and Pyrilamine maleate. The method is UV compatible and can be used as a general approach for analyzing similar compounds.
Acetaminophen (Paracetamol)CaffeineMaleatePyrilaminePyrilamine maleate