Transcript of Mastering LC-MS/MS: Pro Tips for Method Development (LC-MS/MS 101)

hello and welcome to the second session of lcms Ms 101 method development today we're going to focus on strategies and techniques for method development for Mass spectrometry assays I'm Crystal Holt and I'm your moderator for today today I'm joined with our speaker Dr Carl ochin Dr Carl ogin is a senior scientist driving food environmental forensics clinical and cannabis applications at psyx before joining sex he completed his PhD at Colorado School of Mines where his research focused on non-targeted characterization of complex surfactant mixtures including aqueous film forming Foams which led to the discovery of several novel purr and polyfluorinated alkyl substances also known as pfas that since have been found in a variety of environmental samples and Industrial chemicals since joining psyx Carl has worked with numerous Labs creating and implementing both quantitative and qualitative methodologies take it away Carl thank you so much Crystal um so if you're new or just joining us today welcome if you're returning that's awesome glad to have you here and we're going to be talking about method development so method development is really the time that we get to be a little bit creative um as analytical chemists so we're going to cover the approach to making a new quantitation method um so this would be for a new assay um just from scratch but also these same steps could be used to add a compound to an existing NASA which is something we get asked a lot to do so up first we're going to be talking about compound optimization so this is going to be our sort of first thing we need to do we need to Define what our compounds are we need to choose what compounds we want to look for and then we need to optimize for those parameters so the method we'll be moving today will be a quantitative method so this will be a standard mrm method so up first we're going to go ahead and identify our product or do that q1 scan so if you remember from the previous session when we were talking about parents the parent is what we're interested in so let's say if we are interested in resurfing that is what we're going to be looking for so we first need to identify it so we're going to do a q1 scan which is going to scan an area and we're going to talk a little bit about how to set that up next we're going to define the fragments so we talked a lot last time about fragmentation fragmentation methods so those fragments right are sort of like the compound's fingerprints and in an mrm experiment if you remember we're going from a parent to a daughter or a precursor to a fragment so we really need to First Define what fragments exist for that compound and last and probably most important we need to optimize parameters for those specific fragments so let's get started so the first thing we need to decide is what ionization technique we're going to use so lcms right we're mostly talking about electrospray ionization that's the probably the most commonly used ionization technique but within ESI there's positive and there's negative there's also apci so if you are dealing with compounds that are little nonpolar maybe sort of flirt with that GC realm um maybe apci would be a good choice we're going to talk a lot about ESI pretty much exclusively but I like to use this example as a time when hey apci was actually a better choice so for this compound ergonox 1076 I went ahead and ran it um four times um the same instrument with the same column the same standard and I just changed the ionization techniques so I went from an ESI in negative mode to an apci and negative around and I did subsequently in positive mode as well and what you see is you get this really nice giant Peak for apci negative this kind of makes sense for this compound it's got a really long alkyl tail but for ESI you get a much smaller signal and for esight positive um it's not even shown because there really was no signal so we're going to want to take a some time to sort of consider the analyzer we're interested in so what are their size um how polar are they were they traditionally done by gcms before we kind of move into our ionization approaches but for the majority of the time if not more than 95 of the time we're going to be dealing in the ESI Realm so what's the difference between positive and negative and just put very simply the difference between positive and negative would be in positive mode we're really adding something so here we would be adding maybe an ammonium or a sodium and we call these things addicts or plus h which would be the com most common uh addict that you would observe so in positive mode we're adding something to our compound so even if for your compound has an true mass of let's say a hundred if we add a plus h The observed Mass would be 101 right because we're adding something that compared to negative mode where we're taking something away so in negative mode we would be for example removing a hydrogen so we would see a minus one so if that same compound had a mass of 100 our observed Mass would be 99. and that's really why we have to do this q1 scan we need to observe what the mass we want to isolate for our mrm experiment and we have to identify what that mass is so for this example I have a polyethylene glycol and I'm showing you this because we have the plus h and the plus ammonium addicts shown so this would be just our simple q1 skin I knew the mass of this polyethylene glycol and I just scanned from a range of 414 up to somewhere around 440 and what I see is kind of interesting if I were to just go ahead and assume that my plus h was the you know most abundant addict I'd really miss out on this big ammonium Peak so that plus nh4 which has a lot more sensitivity or a lot more intensity Than This plus h so that's where sort of doing these definitions of what I'm actually looking for become really important so with positive mode which is what we'll talk about primarily we'll talk about negative mode a little bit um you'll commonly see plus h plus ammonium plus sodium so we want to keep our eyes peeled for those plus one plus 17 plus 23 of our parent compound versus in negative mode where most of the time we're just looking at a deprotonated compound foreign ER from last session we're building our mrm method so the first thing we need to do is identify our parent in this case we have a parent of 215 so our quadruple is going to filter out everything that's not 215. we then need to go ahead and create some fragmentation so we're going to do that in Q2 or the Collision cell so fragmentation is going to take place and that would be our next step we need to define those fragments what what are those masses that that compound fragments into and then finally our Q3 will be used as a filter again to filter out a specific ion in this example would be that 185 . so that's great um that's a nice little animation of you know what's happening but what are the parameters we're really after um and we're really after these three major parameters the declustrian potential so if you remember that's the ability to or the energy you use to minimize solving clusters so that's really going to depend on the parent Mass um or the parent compound rather the Collision energy so that's that energy we're using to create fragmentation and then the Collision exit cell potential or the cxp so that would be the energy used to transmit ions through Q3 so these are the three things that we are going to optimize for each one of those transitions but we first have to know what we're looking for so I'm going to use the example for a certain resurfing has a mass of 608 so this massive 608 great we're happy what am I going to do how do I how do I decide or how do I identify what to actually isolate or filter on q1 so the experiment I'm going to do is called a just a q1 scan and essentially what we're doing is instead of having that quadrupole fixated on a specific mass we're letting it scan a mass range and typically I like to let that mass range be plus or Plus in the case of positive mode uh 25 Dolphins from whatever my true mass of my compound is and I'm doing that because I want to include all of those addicts so I want to include that plus h that ammonium attic that plus 17 and that sodium adding that plus 23. since those are the more common addicts that I would observe so when I do that for resurfing I see I get a really nice Peak at my plus one so we've protonated this we have 609 now um and that's what I'm going to choose for my subsequent experiments because it's giving me the best intensity so I've identified my parent and the next step is to really figure out what is it going to fragment into so to do that we're doing a product iron scan so we're kind of moving our way down the quadruples as I like to say so we've decided what our q1 is going to look for now we need to get Q2 involved and we got to create some fragments so product iron scan we're going to filter for just that 609 determine that's the best um fast to to filter on it's going to give us the best sensitivity now we got to figure out what fragments does resurfing make so we are going to essentially have our Q3 scan from about 50 Daltons since that's a nice lower limit for a quadrupole up to plus one of our parent Mass so we determined that our observed mass is 609 so I'm going to have Q3 stop at 610 just because I like to see that parent mask in my msms spectrum and it's just the scan that range and we're going to read all the fragments so as we ramp our Collision energy we see fragmentation so we typically will ramp Collision energy right because we don't know what an optimal Collision energy is for any of these fragments yet we haven't done that experiment so instead we're going to ramp it and we're just going to observe what happens and what I like to do is pick the top five fragments it's a little bit of Overkill some would say but usually if you choose five you're in a pretty good place in case one of them has a matrix interference as you kind of move down in your method development stage so it gives you a little bit of wiggle room if you don't have five fragments which happens all the time for example in the case of fluorochemicals typically we only have one maybe two fragments take what you can get and then over five is usually a little bit of a little bit of Overkill but everything we do from this point on isn't going to take any more time if we do it with five fragments or if we do it with two fragments so having extra just gives you a little bit of flexibility in the long run so we've picked our fragments so we're going to do our mrm experiment and we're going to optimize for those parameters we talked about earlier so the first one would be the clustering potential so declustering potential right is the voltage to minimize those solvent clusters so it's before any fragmentation is occurring uh it's right at our source it's what's kind of I like to say pulling our compound in um so because of that all of our mrm transitions shown here should look the same in the sense the profile should look the same so we have those five fragments that shows that 397 the 195 and we have their profiles and we start to see this decrease followed by a little bit of an increase in this sort of level area-ish before it decreases again and that's great just depending on the parent all of these should look exactly the same so if I was going to choose an optimal DP value I'd choose something in this range probably around 150 ish would be a great value for resurfing but why did I bother doing this with all of those fragments if it's going to be the same and it's based on the parent what's the point and the reason I like to do this is kind of a personal preference but I like to use it as a little bit of a forensic tool um so because uh we're looking at fragments of a parent and that parent should react the same way to the clustering potential every time if we see a different profile like the one shown here in pink uh I can go ahead and say hey uh turns out you know maybe maybe I chose a fragment that was really in the mud it was very low in intensity or maybe I have some background ion that's also has a mass of 609 and it's giving me sort of this interference uh or false Peak and I choose that as one of my fragments I can use the clustering potential as sort of a sanity check to make sure hey that profile and pink is very different than the one I saw in blue and because of that I can go ahead and say nope that's not resurfing so I'm not even going to consider it I'm going to remove it or maybe this would be a good time for me to go back and reinfuses for surfing and say hey I'm confident this is resurfing right so it can be a little bit of a tool that you can use to make it so you don't make any mistakes in the long run save yourself a little bit of time so we've written down our declustering potential that's going to be the same for all of our fragments we chose 150. now we're going to go ahead and ramp that Collision energy so when we ramp the Collision energy we're going to ramp it for each one of the mrm transitions or each one of those fragments each of them are an experiment uh individually and typically we'll ramp it somewhere from five volts um up to 150 and when we do that what you usually see is that things with smaller fragments require a little bit higher collusion energy it's not always true but it's a good rule of thumb so as you increase the Collision energy you'll start to see smaller and smaller fragments so I've done that here each one of these represents my five fragments I chose in the earlier slide I can go ahead and choose my ideal Collision energy so for that 195 it'd be about 50 compared to that 174 which may be in the 60 range and I'd go ahead and transfer all these values to a notebook or Excel or however right into my method however you're sort of keeping your notes and finally and last but not least our Collision cell exit potential so this is remember that value that we need for two three and the one thing about this is if we consider all the intensity that you could gain in terms of compound optimization Collision energy into clustering potential are going to account for 95 ish of the intensity on the table compared to cxp which is a pretty small amount typically if you're choosing a value within the default range 10 to 15. um you're going to be almost at an optimal value right from the start and in that case that's what we see here so somewhere between that 10 15 maybe a value of 12 would be the optimal sort of point to give us the most sensitivity um so what I like to tell folks when they're when they're doing compound optimization is if you need that little bit of intensity uh to get to your limit of quantitation or limited detection cxp can help if you're talking about a few percent but if you need more than that going after something like Collision energy you need to clustering potential or some other way to gain that sensitivity is going to be a little bit more fruitful for you and all of this of course can be done automatically so in most softwares like sex OS we have an automatic script that's going to go ahead and just go through this process for you that's great especially if you have a lot of compounds um so you don't have to do it manually which can be a little bit tedious the reason I kind of bring this up is even if you are choosing an automatic workflow which again great um you just want to be a little bit wary since we are using something like a syringe pump to actually introduce the compound into our Mass Spec every once in a while I'm I'm guilty of it I'll leave a bubble behind in my syringe and I'll get a spike in intensity and that can be a little bit confusing sometimes for softwares because they might think hey that spike is actually the most intense Point really it's just an artifact for me not doing a good job getting bubbles out of my syringe so if you do choose one of these automated approaches just go ahead and give it a glance over make sure you're you know still paying attention and using some of the rules we talked about to decide if this data is good data so we set up our compound optimization our next topic we need to cover is chromatography so this is really important so unlike before we now have our LC in the mix so we're no longer just diffusing um from a syringe so we've got a sample that contains our standard and we're going to go ahead and inject it onto our mass spec and when we do this uh our goal here is to go ahead and choose a column we need to choose a mobile phase which includes a modifier and then we like need to use that chromatography you might say use that chromatography I mean optimize it so go ahead and remove interferences or separate our interferences from our lines of Interest get our ideal duration Etc so how much of a difference does a column make so in this example I have ergonox 1135 which I ran on the same instrument with the same mobile phase same sample the only thing I changed was the column and what we see I've got an F5 and a C8 and a c18 for my F5 I had less intensity than my C8 and c18 and with my C8 I had the compound eluding a little bit earlier and I also didn't have some of that tailing that was occurring with the ca team so that's great not a huge difference you could kind of argue between the C8 and c18 you get a similar result um but when we look at a larger compound like ergonox 1076 so the difference here would be that much longer alkane tail going from a C8 to a c18 we see a big difference in retention time so between that CH and that c18 there's several minutes I could optimize my chromatography sure but I have a really nice Peak for my C8 and this might be what you're after you might really want that much separation maybe you have a bunch of polymers you're looking at and you really want them spread out in my scenario when I was doing this experiment that's not what I wanted so I ruled out the F5 just because of that intensity but I ended up choosing that C8 because it gave me enough separation between the analyze I was interested in and then I can kind of shorten up method when I use my chromatography like we'll talk about but what are some of the common sort of starting stationary phases that you can use so when we talk about stationary phases if you remember from the first section we're talking about selectivity and selectivity is really going to impact our chromatic resolution so kind of like what we saw in the earlier slide even the difference between a C8 and c18 led to a big difference in my retention time so which stationary phase you choose it's got to depend a little bit based on your goals but also it's going to be heavily impacted by what compounds you're interested in so if you're doing something like positional isomers something like a biphenyl especially if it has a bunch of rings can be really useful versus if you're doing something a little bit more polar and I should have said earlier I'm really talking about reverse space chromatography that's what I'm going to focus on so I'm not really talking about hillock very much but if you have questions feel free to ask so we're going to be talking about first phase so if I have something polar so bipolar compounds a little earlier right um something like eluded Omega polar is a really nice option this is a c18 but it will retain sort of things like dominicide or acivate some of these really small polar anilines a little bit better than just a traditional CA team but if you're just starting out and you're totally unsure um starting with sort of just the general c18 approach it's a really good starting point you're going to get an idea of what's retaining what's not retaining you can even start to experiment with some mobile phases and then if you need to go ahead and do some optimization on your stationary phase later or you might say this is just acceptable but beyond the stationary phase what other variables can affect micromatography and one would be column length right so as our column gets longer our pressure typically goes up which can be totally fine you'll definitely want to make sure your system your LC system can handle that so if you have just a standard hplc you want to make sure your pressure isn't exorbitantly high as you know 12 000 PSI or something like that where you're going to damage your instrument but usually with column length you're not too worried about that that what comes into play with particle size that being said if you feel that way a couple tricks that you could do to sort of reduce your pressure would be to turn up the column oven so as you increase the temperature in your column oven your pressure should come down the other option obviously would be to go ahead and reduce your flow rate but what else does column like change and the big variable would be the elution time so this might be good or bad depending on your goals so if you are really concerned about speed you want a really quick method you need separation but you want your method to be a few minutes long something like a 50mmoney.com can be a really good choice that being said if you want uh optimal sort of resolution choosing something a little bit longer like 150 millimeter column can be a good choice so if you have a really dirty Matrix um having that resolution might be beneficial because you have a bunch of you know plant metabolites in the sample that you need to separate from your pesticides or other metabolites and besides column length particle size is the other sort of big contributor to our chromatographic resolution so as our particle size decreases our pressure is going to increase so if you want to go with a really really small particle size you should expect to have fairly High pressures so we call that ultra high pressure so those are things like 10 12 000 PSI versus your standard hplc which be operating in the 3000 psi range typically and what's the benefit here so the benefit is as you make your particle size smaller typically you make your Peak widths uh more narrow so if you think of the same amount of area under your Peak but you're making it more narrow it's coming up and up and up so you're increasing uh your intensity or you're sort of making your feet taller separating it from its Baseline so you get a little boost in sensitivity they're obviously going to be trade-offs here one of which I have felt I'd come to you a few times but I'll give you one example of a story so I went ahead and injected well I got a very nice 1.3 micrometer column from the folks at phenomenex they sent it to me and my goal was to look at some pesticides in this really nasty plant Matrix I had doing this on a uh 3.5 micrometer column for a while and everything was going well but I wanted a little increase in sensitivity so I went ahead and conditioned my column and injected my sample onto that column and destroyed that column uh completely destroyed it was never used again couldn't use it um and that happened because I had this really nasty Matrix right uh it was acceptable to use that 3.5 so I had a little bit uh bigger particle size but when I got to that really narrow or tiny particle size I ended up just clogging it pretty much instantly so if you are dealing with sort of these really nasty matrices um whether that be serum or hair or plants um just make sure that you're thinking about your sample prep when you're choosing your column and your column particle size so we've chosen a column let's go ahead and say we chose middle of the road we got to see a team 100 millimeters with a 2.6 particle size micrometer particle size so just straight middle of the road the next thing we need to do is go ahead and choose our mobile phase so when we talk about mobile phase we have our mobile phase a right that aqueous and our mobile Place b or that organic and usually in lcms there's not a whole lot of playing around we do with uh our mobile phase in the sense of a is pretty much always water and B is usually methanol or acetonitrado most Labs would prefer methanol just because it's it's quite a bit cheaper than a seat of nitro but sometimes the ceter nitrile gets you a little bit better resolution where there is a little bit more flexibility would be in your buffers or modifiers so here we have some examples of some common modifiers in positive mode I would say formic acid is almost the most common modifier and we're having modifiers to accomplish a few things one is we want that pH control right um and we want that pH to be stable we don't want to have shifts and pH that are going to cause uh resolution differences or elution time differences so we want that stability and two is we want that pH to be different than the pka of the compounds we're interested in right as the ph and PKA gets very close um if that PKA is the same right we're not really not going to be ionizing efficiently so as we move that away we ionize a little bit better so typically we will go into the two to three PH range for most compounds and positive modes seems to work pretty well one thing you'll also notice here is all of these buffers are volatile so we don't want to use salts things that may be were acceptable with lcuv like a phosphate buffer because we need to do volatilize we're doing a different experiment we need to have things enter the mass back ionize where we're set up different and we're looking at different things we're not doing uh UV analysis anymore um so something like that phosphate buffer would be just inappropriate and if you're unsure um almost always go ahead and just try a generic starting mobile phase first so something like water with some formic acid usually 0.1 percent is a really good starting value and you can add it to your organic as well so something like a 0.1 in your methanol if you are interested in things like ammonium addicts so for example in the slide I showed earlier with that you know huge ammoniumatic Peak adding something like ammonium formate is going to help promote um that addict forming so if you are looking at a modematics adding subliminal morning for me is going to be your best bet and you're going to want to make sure that you're doing this regularly right um these things are volatile so as we let them sit our mobile phase essentially gets we call it old but we're just losing our our modifiers or buffers so if you notice for example the sensitivity of certain ions decreasing over time and you look up on your LC stack and you see that you've been using the same mobile phase for a month now uh it's probably a good time to to make a new batch so we've chosen our mobile phase we're just going to go generic again now it's time to sort of use that chromatography a getter method to meet our standards and the things we need to accomplish so we talked about this a little bit last time so it won't go too far into it but we essentially have these four stages in a chromatogram we have a aqueous stage where we're letting our more polar things uh elute we have this ramp stage and then we have our non-negotiable final two stages be a stage where we clean off the column or we hold it a very high organic so typically this is 95 or above our mobile phase B and we want to hold there long enough to get ideally three column volumes worth of mobile face through our column to really clean that out and finally our equilibration phase which is also non-negotiable we need to get our starting conditions back at the exact same place they were when we did our first injection so we don't have shifts in our attention time and here we would just go ahead and hold for the same three column volumes but how do we use this chromatography because certainly it's not always going to look like a ramp from 95 sometimes we maybe are holding for a little bit to let some separation between some isomers occur um sometimes we might ramp super faster just have an isocratic method so what are we doing when we do that and essentially with reverse phase chromatography as we increase the percent B things are going to lose faster so if we increase that ramping or we increase that starting condition so here I have the same compound the only difference is I have a high starting condition for my percent B versus a low starting condition on the bottom and you'll notice the retention time is very different it's about four minutes different so this could be great if I'm looking for a really fast method um for this specific compound but if I have things that are maybe super polar and I start at a very high organic it just won't be retained by my column at all and it will just kind of fly through the system and I won't have a nice Peak so there's going to be a little bit of a balance and that's like that with everything with method development right we're talking about building a customized method for your analytes so it's going to be very custom and finally once we've kind of identified that yes this is this is my uh chromatography I'm happy with I want to go ahead and move on to my final stage uh the next thing I would encourage you to do is go ahead and schedule that method so scheduling means that you're essentially telling the software in your instrument a time to look for that analyte so you're saying at two minutes is when I or in this case four minutes at around four minutes is where I expect this analyte to show up um and it does great why am I doing this the reason I'm doing this is because if I don't schedule my method and I have a ton of different analytes it's going to be looking for everything all the time and if it's looking for everything all the time it can't do as good of a job similar to to me if I'm multitasking I can't do as good of the job whereas if I'm focused on one thing and a specific time I usually can do a little bit better so you'll see that in your data quality so if you only have one or two main lights maybe a handful of analyze it's less important but if you're getting into the range of 20 30 40 um or you know a few hundred analytes that's where scheduling really becomes not only important it's pretty much mandatory and the difference in the data quality can be seen here so we have the Kink which was unscheduled and the blue which was scheduled and what you'll notice about the pink is that it's essentially a triangle so each one of those dots represents a data point where the instrument actually collected data so we've got this triangle sort of shape and that triangle becomes really difficult to integrate because it's going to change a lot depending where that Apex is so it's going to change depending on where the instrument was what it was looking for at that time compared to our pink and the blue where we have this nice sort of gaussian peak with plenty of points across it so if there's a little bit difference in where those points are it's not really going to change the area underneath my Peak as much as it would if I was just integrating a triangle so typically we're going to aim for somewhere between 10 and 15 points across our Peak that's a really good range to be in and 12 would kind of be that sweet spot uh gold star area and it's really easy to do once you've determined into your chromatography is what you want we've already done all the compound optimization you would just run a standard go ahead copy your attention times and paste them right back into your analytical method um and you're ready to go you have your scheduled mrm method so easy as that and you get a lot of benefits in terms of data quality so are you happy with our chromatography we're happy with our compound up last we have to optimize for our source conditions so the way I'm going to show you how to optimize for Source conditions might be a little bit different than what you're used to um this is my preferred method it's called on column Source parameter optimization um the real difference here is that I'm not infusing anything so in the past maybe you used a we call them split tees but essentially what you're doing is using a syringe pump to infuse your compound of interest and then also using your LC to sort of introduce your mobile phase here what we're doing is we're taking a sample with our standard with all the compounds we're interested in since we know our chromatography and we have them optimized we're just going to inject it and we're going to try basically a ton of different Source conditions and we'll get the optimal one and the reason I like doing this is because when we look at the parameters that we're optimizing it's going to be really dependent on flow rate and mobile phase composition so we talk a lot about our source conditions last time in the sense of what they are but just to kind of briefly reintroduce them we're going to be optimizing for these five parameters primarily our gs1 which is what's going to help us create that nice spray that's going to let us ionize our compound we have our GS2 and our temperature these kind of work in tandem so we want to promote evaporation so we're going to use a little bit of gas to help the evaporation in addition to our temperature obviously to help that and then we're also going to do our curtain gas that's what's keeping our instrument clean so that's that gas coming out from behind our curtain plate that helps keep all those neutrals and other things that we don't want in our Mass back out and then our iron spray voltage so this is the actual voltage we're applying to promote that ionization and since these kind of depend on our flow rates they depend on our mobile phase composition to a point um I like to do it on column and uh one example that I have is I was working with a lab that was doing some cannabis analysis and they did their Source optimization everything looked good they're doing it by just infusing their standard and they had their LC set to 50 50 in terms of 50 water 50 methanol and then when they went to actually run the method what they noticed is they weren't seeing any peaks in the beginning of their chromatogram and they're having really hard time figuring it out so I flew out there and the what was happening was essentially uh kind of easy uh to see because if you looked at the source they were essentially just washing their curtain plate they had set the temperature so low because they're using more organic when they did their optimization that when they did their starting conditions which was 100 water all the water was just rinsing off their curtain plate going into their Source exhaust and causing them a bunch of issues um so just my little example of why I choose to do it this way and I think it's easier too so we're going to go ahead and tell our instrument what we want it to run in the sense we tell it the chromatography method that we've already developed we tell it the compounds it's going to look for with their optimized parameters and then we're just going to choose some generic Source conditions so these will vary a little bit depending on the source that you have but here's just some of my sort of generic starting conditions and really it doesn't make a difference as long as you can see the compounds you're interested in we're going to optimize for these so if you can see the compound you're good to go don't worry about it too much so when we do optimize for these conditions what are we doing we're going to go ahead and inject that sample and we're going to inject it a lot so I like to set this up overnight and we're basically going to put it through the paces and run all the conditions we want so for current gas for example the minimum current test I would run on a method would be about 30 PSI but I'm going to go ahead and inject my standard at 30 PSI I'm going to inject it again at 35 and check it again at 40 injected again at 45 and then plot the data so when I plot the data I should see maybe an increase maybe 45 it gives me the most intensity versus 35 Etc and wouldn't do that for all month variables so in size OS there's a nice little script that will do this for you you can do the same thing in analyst as well but if you have a lot of analytes sometimes I'll just make the methods and just change the variables myself it kind of depends a little bit on your personal preference so here's an example of what that looks like so I have avermectin here and I have different Iron spray voltages so I'm starting at 35 down here and I'm moving up to 5000. and what we see is sort of this increase in area as we increase that voltage so what you're probably thinking is that might be true for avermectin but what if that's not true for something else in your panel if you're looking for more than one thing and that happens that happens a lot uh and what you end up having to do is make compromises so avramekton I like to show because it's very temperature sensitive so it lets a really really low Source temperature compared to something a different pesticides like pyrethrins which like a really warm or hot uh Source temperature so I've kind of got to choose a middle ground and since I struggle with my sensitivity on my avermectin I choose to use a lower Source temperature and those are kind of some of the the hard choices that you're going to have to make when you make your own method but you can plot all of them get an idea choose your best values and then you're good to go so method development whether you're adding a compound or you're building a method from scratch um it it should be pretty straightforward you're following these steps but you can see there's a lot of flexibility in all of those steps there's going to be a lot of choices that you need to make that are going to be specific to your goals um so just like always I encourage you to think about your goals first so if your goal is to have the fastest highest throughput method you're going to make some different choices than if your goal is to have the most resolution so if you are in a government lab where you maybe want to separate isomers for some research purpose you might want to choose a really long method and that's fine versus if you are in a lab and you're running customer samples and you need to turn around and make a profit running another shorter method is going to be more important to you so just ask yourself what your goals are and why you're or what you're trying to accomplish with your method but regardless go ahead plan ahead Matthew development takes time and since we live in a world where there's a bit of Regulation which we we all know once we validate a method it becomes very hard to change so make sure that you're happy with your method from the beginning and taking notes is also my my next biggest uh push so if you have those five fragments for example uh all recorded um in the future when you go to do your chromatography or inject your first Matrix sample and you notice there's a huge interference you haven't wasted any time you can just go back to your notebook or your other method and pull a new transition um so take the notes and take your time during these method development stages and if you are interested in sort of the step-by-step approach if you want to know how to optimize a compound where to click when you click psyx now learninghub is a great tool we also have some courses open now on our sites um so you can come out that's actually what I did a long time ago when I first started doing some cute off analysis uh flew out to a static site and did their training and I really enjoyed it so check out the online Learning Hub if you're looking for those more sort of step-by-step instructions and with that if there are any questions I'd be happy to answer them thank you Carl for sharing that great information and now we will move on to the question and answer session of the presentation we have some questions about Source optimization Carl and you were discussing optimizing The Source what's an approach if you have five or six unique analytes or more analytes with different chemical properties and how do you go about Source optimization uh when looking at different classes in one methodology yeah um it's a great question so when I do Source optimization um regardless if I'm looking at two analytes or if I'm looking at 150 anilines I really take the same approach and I really like that on column approach because I can add everything to my mrm method I know when these things are eluding so as long as I choose sort of generic Source parameters where I can see all of those compounds I'm going to go ahead and step through each one of those sort of different variables gs1 to temperature Etc until I have to find an optimal value but if your question is more how do I make those tough choices um you know sacrificing one's intensity for another I think the real answer is you have to look at the limits that you're trying to hit so what is that lower limit that you want to hit or need to hit if it's regulated and make sure that you're achieving those in those difficult compounds first excellent in the same light of compound optimization when you're doing infusion should you be running mobile phase at the same time or should you be working um purely from infusion standards really good question um and uh that actually has come up quite a bit um with customers in the past so what I typically like to do is when I prepare that standard for Infusion I'll actually use my mobile face to make that dilution so I'll take my neat standard and I'll dilute it into my mobile phase so you can do 50 50 of each if you want or you could just do your organic if it has some formic acid but you're absolutely right you'll want some of that modifier in there to promote that ionization like we talked about excellent the next next question is around why do you perform the source optimization towards the end um you talked about kind of re-optimizing at the end and why do you do it in that specific order yeah um the reason being is that uh that's kind of where it falls right because um for my compound optimization I don't really need any information about Source conditions there's no chromatography um so I can do that first so that's why we choose to do that first and we'll need that uh those compound parameters in order to do a chromatography right um so we'll need to choose our compounds optimize for them Etc and for Source optimization if you're doing the on column approach you'll need both the chromatography and those compound parameters so it's it's kind of just where it falls in the process of things excellent also you've discussed Matrix to interference and suppression how do you go about determining if you're experiencing suppression and if so when do you get indications that you should adjust your chromatography to account for that good question uh great question um and some of the clues so there are a few different Clues um that your experience depression and suppression can happen uh in a few different ways right so one way I've experienced it quite a bit is actually in the source so I add Source suppression and one of the big clues that I'm having an issue with Source suppression is if I run a calibration curve and I have my internal standard in there and as my concentration in my calibration curve increases I'll start to see this decrease in my internal standard over time that's because there's only so much that could be ionized at the same time right in my source um so essentially my internal standard and my compound of Interest or my analyte that I'm I'm actually measuring are competing for that ionization so I get some Source suppression that Source depression can happen with internal standard of course but it could also happen with anything in the Matrix as well so if you're seeing something like that one option is dilution dilution can really help with suppression and Matrix interferences excellent we have some questions around column Choice as well and the first is around does column choice and column chromatography change and impact intensity yeah yep um so kind of like what I showed in the example with uh ergonox 1076 and 1135. um you saw that difference in intensity between the F5 and the C8 and c18 um so it was just not doing as good of a job um selecting for for that compound so you will see differences in intensity for sure and then when we get into things like a particle size um and even column length that's where you can also see changes in intensity so it might be the same stationary phase it might be just to see a team like the other CIA team but if it has a smaller particle size you'll likely see a more sort of narrow intense Peak than a larger particle size excellent so there is a tolerance for retention time do you have any rule of thumb of how you might select or set your retention time tolerances for a methane absolutely um I usually default to the uh sort of old adage of plus or minus 0.1 seconds or 0.1 minutes I'm sorry um so about six seconds um that's usually my acceptable sort of wiggle room in terms of my my Peak actually shifting um but what I might do if I'm setting up a scheduled method is sort of broadened or make that window a little bit bigger than that in case something does happen so if you know maybe this one sample um prep method was a little wonky or something happened something occurred it got really hot in my lab and my mobile phase warmed up a ton and my retention time started to shift for my window that I'm looking at I'll usually allow it to be about 20 seconds excellent the next question is around data points and data quality how many data points do you generally require or suggest to ensure reproducibility and high data quality yeah so 10 to 15 is sort of that good range for points across the peak and then 12 would be the ideal area or the ideal range to be in a deep value and the way we're kind of determining that or the way to determine that would be to go ahead look how wide your Peak is so I'll do easy math because it makes it easier for me if your Peak was 10 seconds across we would want to have our cycle time to be roughly a second so if our cycle time was a second we would get 10 points across our Peak so the amount of analytes you have is kind of been a dictate because I did see that question about 12 volume or 12 time is going to dictate that dwell time so they kind of is it interchangeable play but usually you want to adjust your cycle time accordingly to get enough points across your feet excellent the next question is also in regards to dwell time and how big of an impact does dwell time have on analytical sensitivity and intensity of a given Peak yeah um it can have a big uh impact um so I didn't mention it but typically uh we want to make sure our dwell time in a perfect world somewhere around 50 milliseconds would be great um obviously that's not achievable for everything um but we really don't want to dip too much below sort of 10 milliseconds because when we do we'll start to see sort of variability in our Peak intensity and also just a little bit less intensity so if you think about it from just like a a very high level right we're giving the instrument more time to do something and as we give it more time to do something it does a better job so if we can get into that 50 millisecond range we're in a really nice spot in terms of our data quality excellent we have another one in regards to chromatography optimization and a you know wide panel or large panel so if you have a sample that contains analytes with a wide range of pkas maybe anywhere from three to nine in this specimen um how do you determine what's the right pH to provide Optimum chromatography or what's the strategy for that good question and that's a tough position to be in for sure um it's going to depend a little bit uh I would say what ionization mode you're you're using um so it kind of comes this area of uh what fits best for most um and you can do other things so for when I do polymer analysis for example I will add formic acid and I'll also add a modem formate because I have two goals one I want to promote ionization but two I'm almost always looking at that ammonium addict so I want to make sure there's a nice healthy supply of ammonium in my sample to kind of promote that added for me so that might be an option you can go ahead and try adding two modifiers uh if that's kind of your goal but in general I would say go ahead try something like formic acid which is going to work for the majority of things and kind of assess where you're at if there are a few compound classes that are giving you trouble that's where you might kind of get into that range where you can say hey maybe I'm going to try something different excellent we have a couple of questions about mobile phases phases the first is an all preferable how do you choose between methanol and acetonitrile yeah um the the correct answer would be to try both and see what happens um but if that's not an option there are some advantages of methanol over acetone nitrile methanol one would be less expensive so if you're talking about a cost per sample basis maybe that's where you make your choice um and then typically uh acetonitrile is a Little Bit Stronger so if you're dealing with sort of this more nasty difficult complex matrices acetonite child will do a better about job cleaning off your column and also give you a little bit more resolution I would again like to thank you Carl for the informative presentation and thank you for joining us today we hope you found the presentation informative and we look forward to seeing you at the next lcms 101 session