The
first thing we did was prepare the tea the same way the Amazonian natives do.
We took two pieces of bark and put them in boiling water for five minutes.
After filtering, the samples were ready for the LC-MS. Next we used both the
Diamond Hydride™ and the Bidentate C18™ columns to cover the whole range of
polarity in the sample. In the LC-MS data, we identified various compounds
present in the bark extract. These compounds included rutin, quercetin, isoquercetin,
6‐beta‐O‐2',3'‐dihydrocinamonyl‐12‐hydroxy‐(13)‐15‐en‐16,12‐olide‐18‐cassaneoic acid,
camptothecin, and 9-methyoxy CTP. With further study, one of these compounds
could be identified as being responsible for the medicinal properties of the Brownea grandiceps bark extract. Camptothecin
for instance has been shown to have anti-cancer properties.
Monday, April 20, 2015
Cogent™ Columns —Discovering the Active Ingredients in Traditional Medicines
We received a tree bark sample (Brownea grandiceps) from the jungles of Brazil. They say that a
tea brewed from the bark has medicinal properties, and we hoped to find out more
about which compounds were responsible. If you can determine which one has the
medicinal effects, you could make a pharmaceutical formulation of that compound
or even improve upon its properties using a superior derivative. A good example
is salicylic acid, a natural compound found in some types of tree bark. It has
good analgesic properties but also a number of side effects. This has led to
the development of acetylsalicylic acid, also known as aspirin. Our research
here could lead to a similar useful drug if the active ingredients are determined.
Wednesday, March 25, 2015
Bisphenol A Alternatives – Are they really safer?
Bisphenol A (BPA) is a widely used epoxy resin that has come
under scrutiny in recent years. It is so ubiquitous that it was detected in 93%
of surveyed test subjects over the age of 6. You probably handle BPA-containing
products already on a regular basis, such as receipts from the grocery store,
plastic bottles, and many other sources. In today’s world, exposure to BPA is virtually
unavoidable.
As a compound that mimics the action of estradiol, BPA is an endocrine disruptor. In addition, a wide variety of studies have reported a number of other possible health effects. Some companies that have used BPA in their consumer materials have since replaced it with other bisphenol compounds and labeled the new materials as “BPA-free.” This may lead consumers to believe that these products are now safe, but the fact of the matter is that some of these BPA-substitutes are just as toxic, if not more so.
Two examples of these BPA alternatives are bisphenol F (BPF) and bisphenol S (BPS). They are structurally similar to BPA and hence exhibit similar physical properties, making them ostensibly good BPA substitutes. In terms of health effects however, some reports claim they are actually 100 times worse than BPA [1].
Similarity in structure also means that the compounds may be difficult to distinguish chromatographically. Nevertheless, it will be important to have HPLC methods capable of separating these three bisphenol compounds from each other for analyses of “BPA-free” products. Therefore I investigated such a separation using the Cogent Bidentate C18 2.o™ column. I used reference standards of the three bisphenol compounds and found excellent selectivity amongst them using the column.
I was able to obtain a separation in under five minutes using a simple, premixed isocratic mobile phase. The method uses reversed phase conditions and a formic acid additive, which would be amenable to transfer to LC-MS. In more complex analyses such as plasma testing for bisphenol compounds, LC-MS may be the preferred choice. In any case, the Bidentate C18 2.o™ would be suitable for bisphenol separations in various types of samples. Analyses such as these may become more important once the health effects of the BPA substitutes are studied more in the future.
To read more about the application, Click Here.
Reference:
As a compound that mimics the action of estradiol, BPA is an endocrine disruptor. In addition, a wide variety of studies have reported a number of other possible health effects. Some companies that have used BPA in their consumer materials have since replaced it with other bisphenol compounds and labeled the new materials as “BPA-free.” This may lead consumers to believe that these products are now safe, but the fact of the matter is that some of these BPA-substitutes are just as toxic, if not more so.
Two examples of these BPA alternatives are bisphenol F (BPF) and bisphenol S (BPS). They are structurally similar to BPA and hence exhibit similar physical properties, making them ostensibly good BPA substitutes. In terms of health effects however, some reports claim they are actually 100 times worse than BPA [1].
Similarity in structure also means that the compounds may be difficult to distinguish chromatographically. Nevertheless, it will be important to have HPLC methods capable of separating these three bisphenol compounds from each other for analyses of “BPA-free” products. Therefore I investigated such a separation using the Cogent Bidentate C18 2.o™ column. I used reference standards of the three bisphenol compounds and found excellent selectivity amongst them using the column.
I was able to obtain a separation in under five minutes using a simple, premixed isocratic mobile phase. The method uses reversed phase conditions and a formic acid additive, which would be amenable to transfer to LC-MS. In more complex analyses such as plasma testing for bisphenol compounds, LC-MS may be the preferred choice. In any case, the Bidentate C18 2.o™ would be suitable for bisphenol separations in various types of samples. Analyses such as these may become more important once the health effects of the BPA substitutes are studied more in the future.
To read more about the application, Click Here.
[1] “100 Times the Damage: Avoid at all Costs – BPA, BPF,
BPS ,” Mass Report, http://massreport.com/100-times-the-damage-avoid-at-all-cost-bpa-bpf-bps/,
2014-12-31. Retrieved 2015-03-25.
Monday, March 2, 2015
Before you blame the column…
…Rule out other possibilities first! Problems you can encounter
with peak shape, retention, and so on can often be traced to a component of the
HPLC system. For example, consider the following real-world situation which we encountered
in our laboratory.
We were running an HPLC method for ethylbenzene (0.1mg/mL concentration) with a Cogent UDA™ column and observed some problems with the chromatograms (see Figure A). We noticed that our analyte peak was retained longer and longer after each injection and that the peak became broader. Was this changing chromatographic behavior due to column damage? We know the columns are very stable so we investigated other more likely possibilities first.
Also apparent from Figure A was a second, smaller peak that sometimes would show up in the chromatograms and sometimes would not. As before, we investigated system issues as the possible cause. It turned out that it was a problem with the injector. We were using an injection volume of 1 µL, and when we increased it to 5 µL, the peak was consistently present in every run (Figure B).
We were running an HPLC method for ethylbenzene (0.1mg/mL concentration) with a Cogent UDA™ column and observed some problems with the chromatograms (see Figure A). We noticed that our analyte peak was retained longer and longer after each injection and that the peak became broader. Was this changing chromatographic behavior due to column damage? We know the columns are very stable so we investigated other more likely possibilities first.
Also apparent from Figure A was a second, smaller peak that sometimes would show up in the chromatograms and sometimes would not. As before, we investigated system issues as the possible cause. It turned out that it was a problem with the injector. We were using an injection volume of 1 µL, and when we increased it to 5 µL, the peak was consistently present in every run (Figure B).
So
before you blame the column, be sure to inspect everything else thoroughly:
• Check your mobile
phase. Are the A and B lines connected to the correct inlet ports? Has the
flow rate been calibrated for each line? Is the solvent mixer proportioning
valve functioning accurately? When was the mobile phase prepared and does it
need to be replaced?
• Check your method. Are
you using an appropriate mobile phase/gradient for your compounds? Use an ANP
gradient for polar compounds and a reversed phase one for less polar analytes.
Are you operating at a proper detection wavelength for your compounds’ UV
absorption properties? Before running a sequence, look through your method
settings and make sure someone else who used the instrument before you didn't accidentally change anything.
• Check your sample
prep. Are the analyte concentrations suitable? Ensure that overload of the
column or the detector will not occur at these concentrations and dilute if
necessary. Have your samples been properly filtered to remove particulate
matter from entering the system and creating blockages? Have you performed
percent recovery studies to demonstrate that the analytes are completely
present in the final sample for analysis?
• Check your injector.
Is the needle going down far enough to reach the liquid sample? Is the syringe
aspirating the correct volume of sample?
This is of course not a comprehensive troubleshooting list
but it can give you some idea of the type of things that can malfunction. An
HPLC analysis can be complex and involves many factors so do not start with
blaming the column.
Wednesday, February 18, 2015
Cogent Diol™ column is great for biological samples!
Many of you are analyzing blood samples for applications
like clinical trials of pharmaceuticals, forensics testing, and metabolomics
research. We have had a number of successful separations of these types of
samples with the Diamond Hydride™ in ANP mode. You may be interested in seeing
how the latest addition to our TYPE-C Silica™ line of HPLC columns, the Cogent
Diol 2.o™, would work in these cases. We selected three test solutes that would
be pertinent to blood sample testing: warfarin, hydroxybupropion, and codeine.
Let’s take a look at each:
1) Warfarin: An anti-coagulant used for treatment of thrombosis. It does not have an amine group like the other two, which makes its retention lower. Warfarin does exhibit keto–enol tautomerism and its keto form is ionizable. In this form, it has a pKa of 5.0 due to an acidic hydrogen located between two electron-withdrawing carbonyl groups. Its retention was observed to be the lowest of the three.
1) Warfarin: An anti-coagulant used for treatment of thrombosis. It does not have an amine group like the other two, which makes its retention lower. Warfarin does exhibit keto–enol tautomerism and its keto form is ionizable. In this form, it has a pKa of 5.0 due to an acidic hydrogen located between two electron-withdrawing carbonyl groups. Its retention was observed to be the lowest of the three.
2) Hydroxybupropion:
The active metabolite of bupropion, an antidepressant and smoking cessation
drug. Its main structural feature that influences retention in the ANP mode is
a secondary amine. It eluted next, with baseline separation from codeine.
3) Codeine: An opiate used in many pharmaceutical formulations for
its analgesic or antitussive properties. It has a tertiary amine which makes it
amenable to ANP retention. Therefore it eluted the latest of the three
compounds. The peak shape was highly symmetrical, which can sometimes be
difficult to obtain for amines.
We
spiked the analytes in a real blood sample and separated them by LC-MS. Using
extracted ion chromatograms, interferents from the complex sample matrix could
be eliminated. What you will have left are three sharp, well-separated peaks
corresponding to the analytes.
The data
you can obtain shows how ANP chromatography is not limited to the Diamond
Hydride™ column. Any TYPE-C™ column can be used in the ANP mode. Another
interesting feature we discovered is that acetone can be used instead of
acetonitrile in the mobile phase. Acetone has the advantages to your laboratory
of lower cost as well as lower toxicity.
Click here for more information on this application.
Monday, February 9, 2015
Using Near UHPLC to separate an API from a Prodrug
I found that the Cogent Bidentate C8 2.o™ column is great
for separations of mometasone furoate from its active form. The official USP assay
method calls for an L7 stationary phase, and this column is ideal for such analyses.
The method I used here has some useful features.
First off, the Bidentate C8 2.o™ column has a small average particle size of 2.2µm, leading to high efficiency and rapid analysis; I was able to separate the prodrug, the API, and excipients in the cream matrix in under four minutes. This is a near-UHPLC phase but doesn’t require the specialized instrumentation of smaller particles. Hence you can take advantage of the benefits smaller particle size columns have to offer but without the associated drawbacks of UHPLC.
Another convenient feature is that the method conditions are very easy to set up. No gradients or complicated buffers are required. The mobile phase consisted of only isocratic 50/50 acetonitrile/DI water. You can even find this available premixed from some solvent suppliers (just be sure it’s HPLC grade). The flow rate was 0.3 mL/min so solvent consumption was low.
For sample prep, I weighed 5.0 g of the 0.1% mometasone furoate topical cream in an Erlenmeyer flask with a stirbar. After pipetting 50 mL of methanol, I capped the flask and let it stir for an hour. After filtering with a nylon membrane syringe filter, I had a stock solution that I would use for 1:5 dilutions. I prepared two diluted solutions but used different diluents for each. The first used methanol, and in this case the mometasone furoate prodrug should be present without degradation. This prodrug contains an ester bond which can be hydrolyzed to mometasone, the API. Here I used an acid diluent of 90/10 methanol/1N HCl to catalyze the hydrolysis. You need the methanol component for solubility reasons. Then I heated it in a dry bath at 80 °C. Under these conditions, the active form should be present in the solution.
In the extract using the methanol diluent, I saw a small peak early in the run corresponding to one or more excipients. Aside from that, there was only one other peak in the chromatogram. In a methanol diluent, there will be no conversion to the active form so this peak should be mometasone furoate. However, with the extract using the 90/10 methanol/1N HCl diluent, I saw an extra peak eluting just before the prodrug. There was also a slight decrease in peak height of the prodrug, which indicates some of the mometasone furoate had been converted to the active form.
As for the chromatography, the elution order made sense. An ester is a relatively hydrophobic functional group and therefore we would expect the prodrug to elute later than the active form in reversed phase conditions. Indeed, this is what is observed in the data.
Hence, this column can distinguish between the prodrug and the active form with good resolution and a low run time. This may be useful for various analyses, such as studies investigating the rate at which the prodrug is converted.
Click here to read about the full study and see the chromatograms.
First off, the Bidentate C8 2.o™ column has a small average particle size of 2.2µm, leading to high efficiency and rapid analysis; I was able to separate the prodrug, the API, and excipients in the cream matrix in under four minutes. This is a near-UHPLC phase but doesn’t require the specialized instrumentation of smaller particles. Hence you can take advantage of the benefits smaller particle size columns have to offer but without the associated drawbacks of UHPLC.
Another convenient feature is that the method conditions are very easy to set up. No gradients or complicated buffers are required. The mobile phase consisted of only isocratic 50/50 acetonitrile/DI water. You can even find this available premixed from some solvent suppliers (just be sure it’s HPLC grade). The flow rate was 0.3 mL/min so solvent consumption was low.
For sample prep, I weighed 5.0 g of the 0.1% mometasone furoate topical cream in an Erlenmeyer flask with a stirbar. After pipetting 50 mL of methanol, I capped the flask and let it stir for an hour. After filtering with a nylon membrane syringe filter, I had a stock solution that I would use for 1:5 dilutions. I prepared two diluted solutions but used different diluents for each. The first used methanol, and in this case the mometasone furoate prodrug should be present without degradation. This prodrug contains an ester bond which can be hydrolyzed to mometasone, the API. Here I used an acid diluent of 90/10 methanol/1N HCl to catalyze the hydrolysis. You need the methanol component for solubility reasons. Then I heated it in a dry bath at 80 °C. Under these conditions, the active form should be present in the solution.
In the extract using the methanol diluent, I saw a small peak early in the run corresponding to one or more excipients. Aside from that, there was only one other peak in the chromatogram. In a methanol diluent, there will be no conversion to the active form so this peak should be mometasone furoate. However, with the extract using the 90/10 methanol/1N HCl diluent, I saw an extra peak eluting just before the prodrug. There was also a slight decrease in peak height of the prodrug, which indicates some of the mometasone furoate had been converted to the active form.
As for the chromatography, the elution order made sense. An ester is a relatively hydrophobic functional group and therefore we would expect the prodrug to elute later than the active form in reversed phase conditions. Indeed, this is what is observed in the data.
Hence, this column can distinguish between the prodrug and the active form with good resolution and a low run time. This may be useful for various analyses, such as studies investigating the rate at which the prodrug is converted.
Click here to read about the full study and see the chromatograms.
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