The carryover can come from the needle, needle seat, injection valves or the column. To narrow down the problem, first use various needle wash solvents. if that does not solve the problem. Then it could be the column. At the end of the gradient, come back to the isocratic hold at the percentage when the compounds is shown to stick. Stay for a minute at this isocratic, then go to starting condition. See if the carryover disappears. If the carryover (as seen in blank following ULOQ) is high then column needs to be changed.

Try 50 % DMSO 50 % methanol as the strong wash for the autosampler

Hi Chaitanya

What kind of solvants do you use to clean the injection system between the injections ? I think that asenapine and C° stick to the needle. Try to use a more hydrophilic solvant.

1_make injections with (25%ACN 25%H2O 25%isopropanol 25%Methanol)

2_as good practice after any run inject like 10injections with ACN or MeOH to clean system/column/tubing

First differentiate that is this a carry-over or a late eluting peak, you can use needle wash program to prevent if it is from the injector needle. For low conc samples injected right after high conc samples inject a blank, as a solution to problem.

Try to clean up the sysyem with methanol or acetonitrile and see if this will eleimate the problem

The carryover can come from the needle, needle seat, injection valves or the column. To narrow down the problem, first use various needle wash solvents. if that does not solve the problem. Then it could be the column. At the end of the gradient, come back to the isocratic hold at the percentage when the compounds is shown to stick. Stay for a minute at this isocratic, then go to starting condition. See if the carryover disappears. If the carryover (as seen in blank following ULOQ) is high then column needs to be changed.

Also consider looking for dead volume in the system, esp, downstream of the injector. A poorly fitted connection (tubing not fully inserted when compression fitting tightened) can result in carryover bleed out that take multiple injections to clear. Be especially careful with PEEK tubing and fittings, which can slip apart when tightening. Some vendors, including Waters/Micromass, offer preassembled stainless steel fittings that prevent/minimize dead volume.

First, how do you assess the carry over?. What type of solvent is used for cleaning the needle, what kind of UPLC system have? Because, depend on the system you have, and you need to select the cleaning solvents. For UPLC first generation, in order to prevent contamination, the strong solvent must be a little stronger than the mobile phase and the weak solvent should be the same ratio of mobile phase.

if the carry over disappear following this recomendation, the cleaning solvents are the problem, but if the carry over persist, I recommend clean the UPLC without the column with the next solution: Acetonitrile 25 %, Methanol 25%. 2-propanol 25%, Water 25%, Formic Ácid 0,2 %. Note: Do not pass any cleaning solution in the Mass Spec.

You can run the sequence in the following manner;

1. Aqueous blank

2. ULOQ

3. blank matrix

4. blank matrix

5. blank matrix

6. Aqueous blank

7. blank matrix.

The 1. will tell you if the system is clean. If the repeated injections from 3-5 are not reducing the carryover, and there is no carryover at 6, but you see it again in 7, then it means it is coming from matrix. you need to change gradient, if that fails then MRM. if 3-5 are reducing the carryover then it means it is coming from needle or needle seat.

Carry over maky aries in following situations:

1. From auto sampler (Injector loop, injector port, needle)

Resolution: Use different wash solvent combination as suggested by most of them

2. From HPLC column

Resolution: Play with different type of column chemistries

3. Isocratic Vs Gradient elution

Resolution: Prefer gradient elution than isocratic elution. This will definitely resolve the problem in a greater extand. Try to optimize the gradient program as suggested by "SYED LATEEF from Agilent Technologies.

Thank you all for your valuble suggestions

In addition to the above given procedures, the following may also be helpful. I guess, the UPLC is connected to an electrospray ionization mass spectrometer. The electrospray needle or spray capillary need to be cleaned. The tubings within LC and the tubings connecting LC and mass spectrometer may also need to be cleaned or changed. The solvent bottles, solvent filters, sample vials also need to be periodically cleaned.

Clean up the system by injecting methanol , water for 305 times to ensure the removal of any residues if the analytes.

In addition to all the above suggestions my suggestion is to increase the needle wash volumes like Strong wash and weak wash volumes and number of washes and keep needle wash before injection and after injection and also clean the cone with Methanol:water(0.1% FA) for 30 minutes and then next with pure methanol for 30 min then u may get over the carry over problem..............

This article may provide you or anyone else with some ideas on how to diagnose and prevent carry-over issues in HPLC and LC/MS.

"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; http://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html