Detecting Primary Amines

[Roy Kelvin]

Hi Everyone!

I'm a new grad and out in the working world. Help! I've been given a primary amine with no chromophore. Does anyone have a straightforward method for detecting primary amines by UV. My mobile pase is 20:80 Methanol:water and my column is silica based. Trying to stay away from derivatization. Thanks

[Kostas Petritis]

Is UV the only detector in your disposition?

There is always the possibility of indirect UV detection but I would try other detectors first if available (i.e. ELSD etc). Is your amine hydrophobic enough (i.e. will it be retained in a C18?).

[Roy Kelvin]

Thanks for the response. My compound is indeed hydrophobic and retained on the column. I have thought of Indirect UV but cannot find any good literature on the subject.

Roy

[zompa]

Hi,

I am a new grad too... little experience... Don't take what I'm saying as fine gold!!
Anyway, I have been bothering all our friends with Ibandronate...no chromophore.. the only way to detect it at the UV is to set the detector at 195nm... (or around that wavelenght). There is a tertiary amine in the molecule and that is where the "e" is max for amines a far as I know.
You need to avoid certain conditions if you wanna have a try... but if you are interested there is a post about "detecting at 195".
Hopefullly I have been helpful to you.

[Mark Tracy]

Conductivity detection works well for cationic compounds. The linear range is a bit limited compared to UV, but still good enough for routine QC work. You need to keep the ionic strength of the mobile phase low unless you go for suppressed conductivity.

Also, pulsed amperometry works with excellent sensitivity.

[Uwe Neue]

Indirect UV has not been used in LC in some 20 years or so. You may need to go back for a while to find some publications. There are plenty of publications in CE though.

[ccyting]

For primary amine detection, I believe the post column reaction detector will be one of the best approaches. It is a very sensitive detector and you can do a lot of dilution to eliminate the sample matrix effects. Electrochemical detector is a good approach but not for prolong operation due to electrode surface poison. For post column reaction, you need to worry about the band broadening (extra column effects) due to mixing problem when you use continuous flow injection technique. But the mixing problem can be resolved by using air segmenation of the column eluents before adding the derivatization reagents. For UV detector at 195 nm is another good approach but you need to use extra clean HPLC solvents and water to eliminate the baseline problem. Good luck.

[JMB]

Roy,

You might look at these refs.

A) Alendronate by derivatization

J Pharm Biomed Anal. 1989;7(12):1719-27. Related Articles, Links


The determination of 4-amino-1-hydroxybutane-1,1-diphosphonic acid monosodium salt trihydrate in pharmaceutical dosage forms by high-performance liquid chromatography.

De Marco JD, Biffar SE, Reed DG, Brooks MA.

Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay is reported for the determination of 4-amino-1-hydroxybutane-1, 1-diphosphonic acid (AHBuDP) monosodium salt trihydrate, a new inhibitor of bone resorption. The compound does not demonstrate any intrinsic UV properties and thus pre-column derivatization of the primary amino group of the drug with 9-fluorenylmethyl chloroformate (FMOC) at pH 9 in the presence of sodium citrate is required to facilitate UV detection of the analyte. Excess derivatization reagent is extracted with methylene chloride and an aliquot of the aqueous portion is assayed on a polymeric phase (Hamilton PRP-1) at 35 degrees C by reversed-phase HPLC. A mobile phase of 0.05 M citrate and 0.05 M phosphate buffer (pH 8.0)-acetonitrile-methanol (75:20:5, v/v/v) is utilized with UV detection at 266 nm. Application of the method to the analysis of AHBuDP in I.V. solution, tablet and capsule formulations is presented.

B) Alendronate by indirect UV

J Pharm Biomed Anal. 1994 Aug;12(8):983-91. Related Articles, Links


Determination of bisphosphonate drugs in pharmaceutical dosage formulations by ion chromatography with indirect UV detection.

Tsai EW, Chamberlin SD, Forsyth RJ, Bell C, Ip DP, Brooks MA.

Pharmaceutical Research & Development, Merck Research Laboratories, West Point, PA 19486.

Application of ion chromatography (IC) to the analysis of non-chromophoric bisphosphonate drugs in pharmaceutical dosage formulations is described. The method is based on the use of single-column ion chromatography in conjunction with indirect UV detection that obviates the need for tedious chemical derivatization procedures. Diluted drug samples are chromatographed directly on a Waters IC-Pak HR anion-exchange column with dilute nitric acid (1.6-12 mM) as the mobile phase which exhibits a UV absorption maximum near 220 nm. Analyte detection is monitored by measuring the decrease in absorption of the mobile phase. The IC method has been validated and shown to be precise, accurate, specific and rugged for routine assay. Application of the method to the determination of alendronate sodium tablets, etidronate disodium injectable (which requires an eluent pH control for chromatographic resolution of active drug from chloride ions) and clodronate disodium injectable is presented. The performances of the Waters IC-Pak HR and several equivalent columns are also discussed.

JMB

[Rob Burgess]

What concentrations are you trying to detect down to? Will it be a routine assay? Do you need good accuracy / precision (i.e. % RSD < 2%)

Some options could be: ELSD, CAD, and lastly simple MS detection.