Chromatography Forum

Hello all,

I am doing some method development for Indocyanine Green using LC/MS/MS ESI +

My mobile phases are A) 0.1% Formic Acid in H2O B) ACN

I am using a C18 column 3.5micron, 2x100mm flow rates of 0.7mL/min , gradient, 15uL injection on a 6410.

Based on my initial scouting,Full scan MS2, 10minute run time i failed to see molecular ion.

Are there any suggestions?

I will attempt again, but if not will try using a sodium acetate buffer at lower pH ~5.0.

Try to use 5-10 mM Ammonium Acetate add to mobile phase A and reduce the HPLC flow rate to 0.3-0.5 ml/min, mobile phase start from lower composition of B (lessthan 40%B at 0.5 min) and set collision energy about 20-30 eV then you can find the molecular ion of this compound.

Thank you Kindly for the swift reply,

I am just wondering why the analysis of Indocyanine Green requires addition of Ammonium Acetate buffer and a lower pH ~5.0 as opposed to a traditional solvent such as H20 spiked with 0.1% Formic to aide in ionization in the spray chamber.

Also, I would like to ask your expertise in the area of performing scouting runs for method development.

Our primary analysis is small molecular weight compounds from 200-500D, with a ESI + source

Do you usually perform a longer analysis ~10-20min, Full MS2 Scan (example 100-1000D), with a gradient, and flow rates of 0.5mL/min using our 3.5micron 2-100mm C18?

Could i have any ideas as how to setup an ideal gradient? Tips and Tricks?

I would be interested to hear the replies, Thank you all so much.

60% A, 40% B, held for 2.0min

10%A, 90% B, 2.0-2.5, held for 1.0min

60% A, 40% B, 3.5-4.0min

4min run time.

This gradient was obtained from

Chen CY, Fancher RM, Ruan Q, Marathe P, Rodrigues a D, Yang Z. A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile. Journal of pharmaceutical and biomedical analysis 2008 Jun;47(2):351-9.

I am just trying to replicate and am wondering if there is any way to further optimize.

Thanks

For the positive ESI scan, fragmentor voltage at 240V,cell accelerator voltage at 7, i see 771.5 and 787.5, I failed to see 775or776

Any idea why molecular ion not showing? or even 753 due to loss of sodium? I failed to observe both.

Maybe the problem is the concentration of my pure standard dissolved in mobile phase

currently I am using 2,000ng/mL, which would be fine for MRM, but in the MS2 fullscan is covered by noise?

Maybe i should use a more concentrated standard, such as 5,000ng/mL for determining my parent ion, also maybe i should narrow the scan from 700-850?

Any advice?

Our primary analysis is small molecular weight compounds from 200-500D, with a ESI + source
Do you usually perform a longer analysis ~10-20min, Full MS2 Scan (example 100-1000D), with a gradient, and flow rates of 0.5mL/min using our 3.5micron 2-100mm C18?
Could i have any ideas as how to setup an ideal gradient? Tips and Tricks?

Dear Shemesh,
Yes, I am working on drugs analysis for clinical and forensic toxicology. Also, I use C18 column 100mm x 2.0 mm, 3.0 um for wide ranges of drugs and some special column for a drug(s) that have unique properties different from the others.
For the mobile phase I use A is a 5 mM Ammonium acetate in 0.1% Formic acid (Aqouse Phase) and B is a 0.1% Formic acid in Acetonitrile. The flow rate is constant at 0.3 ml/min and the gradients starting from 10%B to 80%B within 16 min and runtime 25 min totally. The compounds of interests most have molecular between 100-500 amu. with acidic, basic and neutral properties (Common precibed drugs and drugs of abuse) the method can also be detect some pesticides.

I am not expert in HPLC method developments and I think LC/MS(/MS) is easier than HPLC in term of analytes separation becuase peak resolution is not major problem as long as the characteristic ion of the analyte is not same.
Usually, I start from consider an analyte properties like pKa of that compound(s) for select HPLC column, mobile phase and start run with survey gradient at low flow rate, gradually increase composition of organic phase and see the result (e.g. peak shape, tailing?, peak resolution from co-elute m/z, RT)
If you are not satisfied with your result. You can adjust the gradient in each of time periods and also the flow rate increase or decrease depends on the result you need.

Tips.
If your mobile phase is low pH (0.1%Formic) at this low pH, basic compouds are positively chaged and RT may be reduced, acidic compounds are protonated and have increase RT when using reverse phase column.

My way to optimize compounds on 6410.

Use Google. Its good to know chemistry properties and all the info that can be useful ...maybe ion source temperature is to high ?


1) take off this long* 100mm column and use something shorter like 10mm column or capillary that will connect your autosampler valve and nebulizer needle
2) use your original solvents but not as a gradient just isocratic flow 1:1 (if long retention time use ACN>H2O)
3) its good to dilute compound in mobile phase so also 1:1 (ACN:H2O)
4) set up ms2 scan range in the way that will catch the possible ionisation (adducts)
5) if you catch precursore ion - set up proper capillary voltage
6) use your optimization tool in MassHunter "Optimizer" - save a lot of time

When you finish optimization for ms detector start the fun with long column and LC - gradient/flow/temperature etc...

Good luck

p.s.
sorry for my english...very bad

.

Hmm, I still am having difficulty.. I really do not think this is the right peak, however this is the only peak observed..I did change to a 1.8micron , 2.1x50mm C18 column.
The molecular weight of ICG is 775 , minus a sodium ion will be 753 for the protonated species...
I am looking for a 753peak, but do not observe....

This is using an isocratic elution, 60% A , 40% B at 0.2 mL/min and 20uL injection of a 15,000 ng/mL standard prepared in mobile phase.

A= 10mM sodium acetate buffer pH 5.0 adjusted with Formic Acid
B= ACN

Hello.
Did you inject the blank sample also ?
I can see 753.1 ion (not big but still there) could you separate it (EIC) and show in different chromatogram like i show below ?

A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile
Cliff Y. Chen , , R. Marc Fancher, Qian Ruan, Punit Marathe, A. David Rodrigues, Zheng Yang

They use different detector but still source temperature is much lower - 120 not 300

Sometimes its good to start from tail* - set up MRM for your compound based on google info.
Source temperature 200
Cappilary voltage 3k
Fragmentor 100
MRM - 753.1 -> 330 (collision 10)
MRM - 753.1 -> 330 (collision 20)
MRM - 753.1 -> 330 (collision 30)

From your chromatogram. Maybe someone can tell us where did those ions come from.
705 - > 719 -> 733 -> 747 (difference 14)
759 -> 773 -> 787 -> 801 (difference 14)

Hello Pepter,

Thank you Kindly for your swift reply.

Hmm.. It looks like this compound requires a low pH to ionize .

I prepared the 10mM Ammonium Acetate Buffer at pH 3.0 as Mobile phase A) initial 60% , B) ACN 40%

This scan was taken with Fragmentor set at 100V and Cell Accelerator Voltage of 4 and Scan Time of 100.

When performing intial method development under MS2 scan is it best to start with default values of 140V and 7 as well as 500 scan time?

Thanks

Hello.
Great that you solve the problem.
Iam working with pesticides and the start vaules on MS2 Scan: fragmentor 80V and scan time 500 works fine for me.

The "Scan Time" value in MS2 Scan mode or "Dwell time" (on my old Agilent 6410A)
If you set up scan time 500 for one segment (your example : one segment, mass range 600-900)you will get the 2.03 cycle/s or
496.6 ms/cycle. So now when you integrate your TIC peak from chromatogram you will probably get peak width around 0.25 min=15s
it means that you have about 30.45 cycles that catch your peak (30 points). Proper Scan Time/Dwell will give you enough points
for good peak shape and wont generate to much noise (from literature its about 15-20 points for peak).

Now when you have the precursor ion will you use MassHunter Optimizer or you plan to set rest of values by hand* ?

p.s.
your MS detector is 6410 or this new edition 6420/30 with double turbo pomp ?
sorry for my english very bad.

Hi Pepter,

Thanks for the information. I have the 6410 model with single turbo. Actually yesterday I was successful in finding the precursor ion, I did a few changes, as well as a Product Ion Scan of 753.3.

I have determined the precursor to be 753.3 and the product ion of 330.1 , so I setup a MRM scan.

Today I am going to use the optimizer software to find the optimal Fragmentor Voltage and Collision Energy.

I have a rough idea to use 80-160 for Fragmentor Voltage, and 25-40 for Collision Energy. Nevertheless

with my initial 80 FV and 25 CE values for the MRM, I still do observe a very significant Signal to Noise ratio.

Lets see if I can fine tune the method.

Don't know your experience with Optimizer program. When you set up it for optimization process
you need to load base method (in first tab). You probably notice that if you do any changes in method
in Acquisition window you need also reload the method in optimizer...(optimizer have path for method but
keep in mind the original one - it doesn't refresh). You cant set any segments like waste/ms/waste.

Good thing is that all data is saved in normal MassHunter files so you can open it using Qualitative program
to check step by step all the values that optimizer sets up during optimization process. Usually Optimizer give 4 product ions
in results (most abundant ions) sometimes its good to check files maybe there are better products that give smaller
peaks but also very small noise.