trimebutine maleate analysis in plasma with HPLC-UV

[serap g]

Hello

I working with trimebutine maleate in plasma.I use HPLC-UV method which High-performance liquid chromatographic determination of trimebutine and its major metabolite....by Eun-Hee Joo, Woo-Ik Chang, Injoon Oh, Sang-chul Shin. Mobile phase is a mixture
(18:82) of 0.05 M acetate buffer containing 5mM heptansulfonic acid adjusted to pH 5.5 with acetic acid and acetonitrile . I use C18 reversed phase column ( ACE 5um 250x4mm). But detection limit is very low 0.5 mg/L. Iwant to detect 20 ng/ml . What can I do?

[Uwe Neue]

Your description of the problem is incomplete, since we do not know how much sample you have available. There is a difference of 25 between you stated concentration and the concentration that you want to see. Assuming that you have injected 10 microL of your standard, what is wrong with injecting 250 microL of your real sample? Of course, this assumes that you have enough sample available to do that.

Check first, if you have enough analyte mass in your sample to get to the intended limit with the setup that you have. If this is the case, then one can solve the problem by working with injection volume or with a sample preconcentration (and cleanup) step with SPE. If you are still off, you may consider a smaller ID column.

[serap g]

Hello Uwe
Thank you for your answer. You are right that I couldn`t explain my problem. My english is not good…Our study is based on to determine the concentration of trimebutine in human plasma, is about 20 ng/ml after the oral dose 48 hour later. Just now, we did not begin the extraction process but in fact I couldn`t detected the 20 ng/ml trimebutine standart as 20 ng/ml concentration when I injected 10 ul volume of this standart to HPLC. When I`ve injected 10 µl 0.5 µg/ml trimebutine Standard in mobile phase, peak are is found about only 5,5. with column or without column. So that; to beginthe study in human plasma, we must determine the accurate adopted conditions. I prepared the stock solution of trimebutine in metanol. All the dilutiond for the standart are prepared in mobil phase. I`ve defined the stoichiometric composition of mobile phase before.( 18/82, buffer/ACN). I use the UV detector at 267 nm wavelenght. I`ve studied with different compositions ( valves) of mobil phase in this method. The detection limit value did not change. The temperature I`ve studied at 400 C but I couldn`t find suitable results. The enjection value is almost 50 µl. For this method the referans literature is ‘’high-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma. Eun-Hee Joo, Woo-Ik Chang, Injoon Oh, Sang-Chul shin, Han-Kwang Na, Yong-Bok Lee Journal of Chromatography B, 723 (1999)
Thanks

[Ann]

Hello, I can sympathise with your problem as have experienced it myself (several times, seem to be constantly having to work near the assay LOQ, sigh!)

As Uwe says, things you can do to increase assay sensitivity are to;
1) Concentrate your sample during extraction
2) Use a micro-bore column (e.g. 1mm i.d.)
3) Inject more sample on to the column

The concentration factor that you can achieve will be dependent on how much blood you can take from your patients/volunteers (which will in turn be dependent on how many samples you want to take and the type of patient (baby, child, adult)). Also, it will be influenced by the recovery of your compound as you may find that recovery decreases as you attempt to concentrate your sample more, so you will have to experiment to find the optimal conditions. You also have to consider your required injection volume at this point; you will need to allow for at least two injections for analysis (preferably more in case of problems) and wastage due to the injection process. Obviously, the more you concentrate, the smaller your total sample volume will be.

Micro-bore columns are an excellent way to achieve a fairly large increase in sensitivity. However, you will need to scale your flow rate accordingly and may need to buy a special micro-flow pump if you don't already have one. Typical flow rate for a 1mm bore column is only 0.05mL/min.

As for your detector, there are a few things to consider here too.
Is it performing optimally? (is the lamp old? Has the wavelength calibration been checked lately?)
Flow-cell - are you able to replace the flow cell to enhance sensitivity (go for small volume but longer pathlength)
Are you working at the lamba max (I know the paper you've referenced says 267nm but have you checked this on your system)?

First work out what your current LOD/LOQ is from simple injections of standard solutions, then you'll be in a better position to judge what measures you need to take to achieve your target LOQ.

Best of luck, Ann

[Uwe Neue]

Ann has pointed out already a few things. Please look at this carefully!

Let me approach it from the other side! Assume that you can take a 5 mL sample from the human volunteer or patient. Then you have 100 ng of analyte available. With sample preparation techniques, you can concentrate this to a volume of maybe 50 microL. This gives you a concentration of about 2 microg/mL. You have found a peak injecting 10 microL of a 0.5 microg/mL standard. So you will be fine. At least you are in the ballpark.

Ann mentioned that you may be better off with a smaller i.d. column. I agree and a 2 mm column can give you another factor of 4 in sensitivity. Then your patient may not have to bleed 5 mL, but 1 mL might be OK

Sample preparation will be important. I can give some advice there if you contact me. I have written a book chapter on sample preparation for blood samples such as yours.

[mbulentcakar]

Hi all,
I had a dialogue with Serap off forum. I'll try to summarize her new efforts (on her desire):

-checking detector lamp and finding that it had been working for 6236hrs!! (maybe 623.6?) so she tried a DAD and got 2 times better response. but this is way far from being adequate for her.

-she also tried a UV spectrum and confirmed that the wavelength is correct and 267nm is ideal. also states that reference standard for trimebutin is 99% certified.

-for preventing a possible poor injection, she supplies 10 times more volume of the sample than the vial's specified minimum sample volume.

-approx. 80% recovery from extractions of 0.5, 0.7 and 1.0 mg/L spikes.

She also says that she could only double the sample size.

My advice was on detector and if possible changing the flowcell on Ann's advice. Also, some (much) higher efficiency column may give some better response giving a slim and tall peak.

2 times from detector, 2 times from sample size (maybe 4 combined with higher injection vol) gets the loq nearer to desired value.

On the other hand the literature value should have been obtained with a more or less comparable system. I can't fully explain the high gap (25 times) for the LOQ.

[Ann]

I have also struggled to achieve the quoted LOD/LOQ in published methods, even when using more modern (and supposedly more sensitive) equipment, it's one of life's mysteries!

Definitely worth considering switching to a narrow bore column if you are currently working with a 4.6mm. Even a 2 or 3mm column will help a lot(and won't require such low flow rate as a 1mm bore column and hence, should be achievable with a standard pump).

When you say 'sample size', do you mean blood sample volume or injection volume? What is the current concentration factor during extraction?

All best wishes, Ann

[Uwe Neue]

We are getting a bit closer to understanding the problem, but it still is not complete.

What is the volume of the human plasma that you start with, and what is the sample concentration in this volume?

Once we understand that, we can design the remainder of the technique from sample preparation to HPLC analysis around this information.

[serap g]

Hello Uwe and Ann

Thanks for your ideas. There will be works about this the next days. I will inform you about the progress.
See you.

[DR]

Also -
Do not overlook the extra column effects between the column & the detector - make sure you have as short a length of the narrowest bore tubing you can find between them and make sure everything is properly seated (no dead spaces).

[Uwe Neue]

I looked at the published method. Nova-Pak C18, the column that was used in this method, is likely to give much less retention than your column. This is good for a factor of 2 in the sensitivity. You may be able to play with the solvent composition to get there. The lowest concetration reported was 10 mg/mL of plasma which was concentrated 5:1 in the sample preparation process, for a final concentration in the injector vial of 50 ng/mL

There is still a roughly factor of 5 difference between where you are and where they were, assuming that you can adjust the method to a lower retention. You may be able to get another factor of 2 to 3 from working with the detector setting of your instrument. Since you got 10x more sample than you inject, you could get some gains by injecting more.

So it is not impossible.

[tom jupille]

Also check things like your detector baseline noise (age of the lamp; time constant or data aquisition rate;degassing). Since detection limit is based on signal/noise ratio, every bit of noise reduction reduces the detection limit. I know that I sometimes focus so much on separation chemistry that I overlook some of the simple things.