RT Variation

[sankie]

The following is the method I am using for analyzing Impurity F by HPLC.

Impurity F (by HPLC):

(Reference: General method 2.2.29 Liquid chromatography and product monograph 0931 as per Ph.Eur.)

Derivatisation solution:Prepare the solution immediately before use. Dissolve 250 µL of fluorodinitrobenzene in 25.0 mL of acetonitrile (HPLC grade).

Blank solution:To 5.0 mL of acetonitrile (HPLC grade)add 100 µL of triethylamine and 1.0 mL of the derivatisation solution. Shake well and heat at 60 °C for 30 min. After cooling dilute to 10.0 mL with acetonitrile (HPLC grade).

Test solution:

Prepare the solution immediately before use. Suspend 10.0 mg of the substance to be examined in 5.0 mL of acetonitrile (HPLC grade)and sonicate for 5 min. Add 100 µL of triethylamine and 1.0 mL of the derivatisation solution. Shake well and heat at 60 °C for 30 minutes. After cooling dilute to 10.0 mL with acetonitrile (HPLC grade). Filter, or centrifuge at 800 g for 5 min before use.

Reference solution:

Dissolve 1.0 mL of Metformin impurity F WS (corresponding to 36 mg of Dimethylamine Hydrochloride or 20.0 mg of dimethylamine base) in 100.0 mL of acetonitrile HPLC grade. Dilute 2.5 mL of the solution to 100.0 mL with acetonitrile HPLC grade. To 1.0 mL of this solution add successively 5.0 mL of acetonitrile HPLC grade, 100 µL of triethylamine and 1.0 mL of the derivatisation solution. Shake well and heat at 60 °C for 30 min. After cooling dilute to 10.0 mL with acetonitrile HPLC grade.

Column:

- Size: l = 0.125 m, Ø = 3 mm
- Stationary phase: spherical end-capped octadecylsilyl silica gel for chromatography R1 (5µm)Column: Merck LiChroCART Purospher® RP18e 125-3mm, 5µ column
- Temperature: 30 °C (if column oven is available)

Mobile phase:

- Mobile phase A: phosphoric acid R, water R (0.1:99.9 V/V);
- Mobile phase B: acetonitrile HPLC grade

Mobile phase gradient:

Time (min) Mobile phase A (per cent V/V) Mobile phase B (per cent V/V)
0 60 40
0-10 60 45
40 55

10-11 45 25
55 75

11-15 25 75


Flow rate: 0.7 mL/min.
Detection: Spectrophotometer at 380 nm.
Injection: 5 µL.

Identification of impurities: use the chromatograms obtained with the blank solution and the reference solution to identify the peak due to the impurity F derivative.

Retention time: Impurity F derivative = Between 5 and 6 minutes

System suitability: From reference solution:Resolution to beminimum 3.0 between the peak due to the impurity F derivative and the nearby eluting peaks due to the derivatisation reagent.

Specification: Impurity F:Not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.05 %).

Now The problem is, i am getting RTshift in impurity F RT and the limit is 5-6 it usually goes above six.

Can you please let me know what I can do?

[tom jupille]

Questions:

1. Is the shift gradual or abrupt (i.e., did the retention time gradually get longer or did it jump all at once to the higher value)
2. Is the shift systematic or random (i.e., does it bounce around?)

Tests:
1. Run the dwell volume test as described here: http://www.lcresources.com/resources/TSWiz/hs410.htm , but look at the linearity of the gradient. If you see any non-linear regions, that indicates that your proportioning system is malfunctioning.
2. To confirm, run the step-test as described here: http://www.lcresources.com/resources/TSWiz/hs450.htm

If the proportioning system checks out OK, and the retention time shift is gradual, that suggests that the problem is an aging column. Easiest/cheapest fix is to replace the column. If the shift is random, I would look at temperature control as a possible problem.

[Blazer]

Hello,

First, have you ever gotten this to work correctly? Meaning, is this a recent change or is this your first time trying to run this test and it's not working?

Next, let's make sure you're working with the current version of the Ph. Eur. monograph. I ask because when I look at the Ph. Eur. online, I see this monograph and the retention time for Impurity F is listed as "about 4 minutes". You may find the answer there.

The monograph you describe shows that the column heater is optional, but the version I see online doesn't indicate that. Are you using a column heater? If not, what is your lab temperature?

Does your instrument have a lot of extra-column volume that may affect your retention time?

[sankie]

It is not continuous but it happens around 20 days once.

We are not using column heater and our lab temperature is around 28 to 32

we are running this method from 2012 and this problem is coming recently

[tom jupille]

If it happened once and then the retention time returned to normal, that suggests something like a mobile phase preparation error or a transient failure of the pump/proportioning system (air bubble? sticking check valve?) or an anomalously hot day.

Have you verified the proportioning accuracy?