Agilent 6475 Questions

[Ostravaczech]

In February, we got a new LCMSMS system from Agilent - 1290 and 6475, MassHunter 12.1. The system is mainly used for analysis of “compounds of potential concern, CPC” (hormones, personal care products, pharmaceuticals), and PFAS by EPA 533. Both applications are SPE enrichment, 0.5L to 1 mL for CPC, 0.25L to 1 mL for PFAS. Here are my questions:

1. Autotune only? No manual tune? I talked to the app chemist at Agilent and he says I should be able to see manual tune. I do not seem to have any options or tabs that will point me to the manual tune.

2. In the ESI negative, in order to meet MRL of 1 ppt for some of the hormones, I am at gain 10 which is the highest gain allowed while using 20 uL injection. Is this normal?

3. While using polarity switching, with gain 0.2 for ESI(+) and 10 for ESI(-), I am not able to produce decent curves, all are convex. When I split the method to 2 separate ones, the ESI(+) curves look great, ESI(-) not so much.

I sent data to Agilent this week but the app chemist I talked to really didn’t have any solutions except "clean the capillary and retune”, so I will be talking to someone else next week. In the meantime any chemist out there who has seen similar issues and may have some answers and can help?

Thank you!

[DReggio]

1. Agilent is disabling user manual tune on new equipment. It is locked behind a service account that they are not allowed to give you the password for.

It's a really unfortunate decision that has turned my off of Agilent mass specs. Their instrument will now require recalibration when previously a minor tune adjustment could keep it running.

I have not been able to get an answer from Agilent as to why they have locked users out of this essential functionality.

[James_Ball]

Sounds like it might be beneficial to keep on hand an older version of Mass Hunter.

They did this to the ICP/MS several years ago, taking away some very useful troubleshooting items.

Agilent has been our go to for most instruments for decades, but now that they are becoming like all the rest maybe time to look at other vendors.

As for the sensitivity, it will normally be lower for negative ESI than for positive. We had a 6495 for PFAS a few years ago and it began super sensitive, then all of a sudden I lost almost 80% area counts. Had to call Agilent in and they replaced I believe the collision gas valve and it came back. If it is still in warranty definitely have them come look at it just in case.

[bcd_GCLCMSMS]

In February, we got a new LCMSMS system from Agilent - 1290 and 6475, MassHunter 12.1. The system is mainly used for analysis of “compounds of potential concern, CPC” (hormones, personal care products, pharmaceuticals), and PFAS by EPA 533. Both applications are SPE enrichment, 0.5L to 1 mL for CPC, 0.25L to 1 mL for PFAS. Here are my questions:

1. Autotune only? No manual tune? I talked to the app chemist at Agilent and he says I should be able to see manual tune. I do not seem to have any options or tabs that will point me to the manual tune.

2. In the ESI negative, in order to meet MRL of 1 ppt for some of the hormones, I am at gain 10 which is the highest gain allowed while using 20 uL injection. Is this normal?

3. While using polarity switching, with gain 0.2 for ESI(+) and 10 for ESI(-), I am not able to produce decent curves, all are convex. When I split the method to 2 separate ones, the ESI(+) curves look great, ESI(-) not so much.

I sent data to Agilent this week but the app chemist I talked to really didn’t have any solutions except "clean the capillary and retune”, so I will be talking to someone else next week. In the meantime any chemist out there who has seen similar issues and may have some answers and can help?

Thank you!

1. If autotune passes specifications, that should be adequate.

2. Wow! 20 uL injection is large. What diameter/type of chromatography column are you using?

3. Polarity switching never gives me good quantitative results for all analytes. I believe it is difficult to get enough "accurate" points across a peak with switching, plus there is more instability. Unfortunately, you may have to separate methods and run the sample twice. The instrument front end needs to be really clean for PFAS to produce linear curves. If the curve is not too bad and the method allows it, you may try: decreasing the curve concentration range at the high end and/or apply quadratic fitting with a weighting of 1/x (or possibly 1/x^2).

We also tried running aqueous 1 mM ammonium fluoride as a mobile phase modifier for hormones as recommended by Agilent in a technical note (5994-0317EN) with some luck.

[Ostravaczech]

Sounds like it might be beneficial to keep on hand an older version of Mass Hunter.

They did this to the ICP/MS several years ago, taking away some very useful troubleshooting items.

Agilent has been our go to for most instruments for decades, but now that they are becoming like all the rest maybe time to look at other vendors.

As for the sensitivity, it will normally be lower for negative ESI than for positive. We had a 6495 for PFAS a few years ago and it began super sensitive, then all of a sudden I lost almost 80% area counts. Had to call Agilent in and they replaced I believe the collision gas valve and it came back. If it is still in warranty definitely have them come look at it just in case.

I come from many years (15+) with Qtrap 4000 which was a great machine. Even though I had to run 2 separate methods, I never experienced issues with linearity or sensitivity. Sadly, the instrument was replaced with 6475 which we piggybacked off of another government entity here in town so wouldn’t have to go through RFP. I did place a couple of service calls and so far they have not been able tom give us satisfactory answers. Anyway, we use this instrument for EPA 533 - so far so good, and few non-compliance monitoring for advanced water treatment (direct potable reuse).

[Ostravaczech]

In February, we got a new LCMSMS system from Agilent - 1290 and 6475, MassHunter 12.1. The system is mainly used for analysis of “compounds of potential concern, CPC” (hormones, personal care products, pharmaceuticals), and PFAS by EPA 533. Both applications are SPE enrichment, 0.5L to 1 mL for CPC, 0.25L to 1 mL for PFAS. Here are my questions:

1. Autotune only? No manual tune? I talked to the app chemist at Agilent and he says I should be able to see manual tune. I do not seem to have any options or tabs that will point me to the manual tune.

2. In the ESI negative, in order to meet MRL of 1 ppt for some of the hormones, I am at gain 10 which is the highest gain allowed while using 20 uL injection. Is this normal?

3. While using polarity switching, with gain 0.2 for ESI(+) and 10 for ESI(-), I am not able to produce decent curves, all are convex. When I split the method to 2 separate ones, the ESI(+) curves look great, ESI(-) not so much.

I sent data to Agilent this week but the app chemist I talked to really didn’t have any solutions except "clean the capillary and retune”, so I will be talking to someone else next week. In the meantime any chemist out there who has seen similar issues and may have some answers and can help?

Thank you!

1. If autotune passes specifications, that should be adequate.

2. Wow! 20 uL injection is large. What diameter/type of chromatography column are you using?

3. Polarity switching never gives me good quantitative results for all analytes. I believe it is difficult to get enough "accurate" points across a peak with switching, plus there is more instability. Unfortunately, you may have to separate methods and run the sample twice. The instrument front end needs to be really clean for PFAS to produce linear curves. If the curve is not too bad and the method allows it, you may try: decreasing the curve concentration range at the high end and/or apply quadratic fitting with a weighting of 1/x (or possibly 1/x^2).

We also tried running aqueous 1 mM ammonium fluoride as a mobile phase modifier for hormones as recommended by Agilent in a technical note (5994-0317EN) with some luck.

1. Autotune passes every time. Instrument is about 10 months old. During the this time, I cleaned the cone / capillary few times.
2. The column is Agilent’s RRHD C18, 100x2.1mm, 1.8 um. Flow 0.4 mL/min, ESI+ buffer is 2.5mM NH4 acetate with some FA to pH~3 and methanol gradient, 4uL inj with gain at 1 gives great sensitivity, ESI- less than 1 mM NH4F and ACN grad, 20uL injection and gain at 10 - sensitivity OK for some but barely seeing 0.5 ng/L for ethynylestradiol. We monitor hormones and some other compounds of potential concern during advanced water treatment for direct potable reuse so those methods have been developed in-house and are not regulated. Curve range is 0.5 - 100 ng/L (0.25 - 50 ng/mL in vial, 500 mL->1mL extraction)
3. I tried polarity switching but reverted back to 2 separate methods (see 1. and 2.) due to really terrible results. The curves are weighted 1/c for all my methods. Cycle time ~300ms, dMRM, resulting in about 25 points / peak.

We also run EPA 533 with success. That said, our MRL currently is set at 2 ng/L.

I come from 15+ years with Qtrap4000 where, even though I was running 2 separate methods, never saw sensitivity or linearity issues. I have been running the methods since 2008 or so….

Thank you for your suggestions.