Results and how the get affected

[Ivymike]

Hello there,
Had a question with regards to results on my Chromatography results

I'm using Shimadzu LC 20AT LPG pump with SIL-20HT Autosampler with SPD-M20A PhotoDiode Array detector with CTO-20 Column Thermostat and CBM-20 Controller

I have a Sunscreen method that runs Isocratic based on following conditions

Column : Kinetex Phenomenex column 2.6 micro PFP 100 x 4.6mm
Column Temperature: 45 degrees C
Mobil Phase: 60 ACN: 40 Water
Flow Rate : 1 ml/min at 313 nm
Back Pressure: Steady at 98 Bars
Injection: 10 microliter

Im running 3 sunscreens
Octylmethoxycinnamate or Octionoxate
Avobenzone
Octisalate

Here is the problem
I'm developing this method to get all the peaks in ten minute run
for the entire last week, I ran couple of sequences and I get near to perfect calibration- I'm running 5 point calibration
But my results do change- ( I'm making the same sample amount )

Monday - I had Octinoxate and Avobenzone at right amount but the Octislate was low
Octinoxate should be about 6% and Aveobenzone is about 2% and Octisalate must be around 4.5%
I got close results to two of the analytes - by trying to dissolve the samples in 50/50 Acetonitrile -Water- It worked for two of the analytes but not the third one( Octisalate)

The standard prep is in 9:1 Ratio Methanol: Water. I can't make my standards in 60:40 ACN: Water simply because the standards are not dissolving correctly- So, I though there must be something wrong with my sample prep.

I took a different approach
I dissolved the samples first in 1 part Water and then QS them with 4 part MeOH to the mark.
Now Im getting the Avobenzone and Octisalate in right amounts and Octionxate in 7% plus region.
Please note, that I have perfect calibration and I beleive my sample amounts are correct as well
Here is how I made the first set of samples and Octisalate was out of range

I dissolved them in 50:50 Water/ Acetonitrile and vortexed the samples in 25 ml vol flask. after seating down for some time I did a 1/10 dilution ( 50:50 ACN:Water) and injected them

The second way how I prepared the samples was
I dissolved them in Water first (5 ml) and then vortexed the samples to disperse them thoroughly- After that I QS the samples with 20 ml Methanol in 25 ml flask and vortexed them again very good , so there will not be any undissolved samples left.
And proceeded with 1/10 dilution
The second set of dilutions were with 9:1 ratio Methanol: Water for the 10 ml flasks and I injected them
And this time Octisalate and Avobenzone were in range but Octionxate was out of the range reading around 7.0%

At this point I'm suspicious of my sample preparation. I know I should make my samples just like standards , but sometimes this doesn't help with the method.
Retention times are coming fine with the Methanol: Water mixes
Standard Retention Times with (60:40 ACn: Water) are as follows
Octinoxate at 4.44 minutes
Octisalate at 5.39 minutes
Avobenzone at 6.5 minutes

Samples with matter of seconds are back and forth- but no significant shift in retention times.
From what I know and read, I know that I have to make my samples weaker than mobile phase- I'm not following that , I know- But I have a problem with dissolving all the standards. SO, thats why Im dissolving my standards in 9:1 MeOH: Water solution and my samples in 8:2 Methanol: Water solution.

If you guys have any ideas please share ;
Thanks in Advance

[Consumer Products Guy]

This is the Consumer Products Guy. I have that name for a reason.

Make up your standards in N,N-dimethylformamide (DMF). Also make up your samples in DMF. For sunscreens, there is a lot of absorbance, and a lot of specificity at your wavelength, keep the concentratins low. And 5 microliter injection is typically plenty.

Although I personally wouldn't use that mobile phase (I'd add a little acid for mobile phase pH adjustment), your mixture is not as complex as others I've worked with, so no need to change anything there if the chromatography meets your needs.