Hello there,
This is the 3rd column we use, when the column is new, after the calibrations are done, the peaks expand as seen in the appendix after 3 days of sample execution, and there is a peak deterioration like a noise and impurity before. We bought HPLC grade water, all liquids are at hplc grade and filtered. However, we could not solve the peak expansion problem.

I wrote about the method and materials we use below. Although I dissolve the samples well and pass them through a 0.22 filter, we are having a problem about quick colon blockage. retention time can be change just 3-5 hplc run. We change our mediums and also filtered every time, prepare medium for fresh. Although we tried many ways to solve this problem, we could not overcome the problem. No matter what we do to overcome our problems like long conditioning, long column wash, we cant achieved it.



Column: Leapsil 2.7 μm C18, 150 x 4.6mm DİKMA firm.
Mobile Phase: Acetonitrile and Hplc grade Water in separate bottles; Working as a gradient.
The hormones in tablet forms are dissolved in 100% methanol (between 1 and 5 ml) and sonicated 5-10 min, after sonification supernatans were taken and filtered. After the filtering, samples added to the vials.
The hormones in liquid forms are dissolved in 100% methanol (between 1 and 5 ml) and after filtering process samples are added to the vials(1,5ml).

Devices: Thermo scientific separation, an autosampler, a DAD detector, it is being studied at 205,237 and 287 nm wavelengths. Each samples has unique retention time and wavelength.
The calibration curve was prepared at 4 points from concentrations of 25, 50, 75, 100 ppm.

The substances we analyze:
1. Boldenone Undecylenate
2.Drostanolone Propionate
3.Methandienone
4. Methenolone Enanthate
5.Nandrolone Decanoate
6.Nandrolone Phenylpropionate
7.Oxandrolone
8.Oxymetholone
9.Stanozolol
10. Testosterone Cypionate
11. Testosterone Enanthate
12. Testosterone Propionate
13. Trenbolone Acetate
14. Trenbolone Enanthate
15. Trenbolone Hexahydrobenzylcarbonate

I am open to any suggestions.

Thank you!