Identifying amlodipine using HPLC

[mashi]

How can i make the peak for amlodipine besylate appear using HPLC?
i'm still new in HPLC. I'm trying to analyze amlodipine at the concentrations of 100 ug/ml to 1000 ug/ml.
here are some additional information bout the HPLC that i'm using:

column: C 18 46-150 mm
wavelength is set at 240 - 362
running time: 1 hour
mobile phase: ACN + KH2PO4 (37:63 V/V)
pH of buffer: 3.5
injection volume: 100 µL

i've tried using 10 mg/ml diluting it in the mobile phase and the peak appeared at 6.34 mins but i can't get it to appear when i dilute it in MeOH to the concentrations that i've mentioned above. Can somebody help me pls?

[JA]

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Hello mashi,

Assuming that you simply desire a method to chromatograph amlodipine with reasonable retention, there are quite a few hits to be gained via a simple google search for "amlodipine HPLC".

I noticed one mention for the use of a 10 mM monobasic phosphate 'buffer' at pH 3.5 (a bit further from pKa1 than I would use) on a Nucleosil C8 column. Your exact column is unspecified, and this is important as there is a reasonable range of selectivity to be discovered with the numerous reversed-phase columns on offer. Not all C8's or C18's are made equal, hence why everybody is selling their own unique version. Other references indicate less retentive column chemistries such as cyano.

With respect to your lack of peak, it is possible your particular column is too retentive for amlodipine with the specified conditions. This is also possible due to your adoption of the literature mobile phase conditions without using the defined column (sorry if I have missed another literature reference which gives precedence to the column you are using).

Generally speaking, detecting an analyte with a reasonable chromaphore such as amlodipine should be straightforward in the range 100-1000 ug/ml with a 10 ul injection. Are you convinced that the peak observed at 6.34 mins with the 10 mg/ml sample is actually amlodipine - do you use MS detection or have an authentic reference sample? You're going to be expecting two peaks with the benzenesulfonate eluting first by reversed-phase.

I would consider adjusting your mobile phase pH somewhere in the region 2.5-3.0 and repeating the analysis at 10 ul of 1000 ug/ml using gradient elution (5-95% acetonitrile) over a shorter period of time. Other suggestions could be made if you elaborate on the particular aim of your work.

[mashi]

Some additional info regarding the column that i'm using:

ZORBAX SB-C8
5 um
4.6 x 150mm

[JA]

I'll reiterate my last post with some data from the USP column comparison site (thanks Tom!)



I'm going to stick with my guess that your column is too retentive under identical conditions. I'm not 100% on this, however, as your column is listed as having more hydrophobic character but is a weaker cation exchanger; both of which could be playing a part in the retention of amlodipine.

Worthy of mention is the fact there is only one column in their list with F < 5. It's lucky you didn't pick the regular Zorbax C8 because it's a mile off