Creation of Humic Acid Columns for Analysis of Drug Binding and Bioavailability in Water

Sazia Iftekhar Author

04/01/2021 Added

26 Plays

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This study focuses on creating a column for liquid chromatography that contains commercial humic acid as the binding agent to characterize the binding interactions of drugs with humic acid using high-performance affinity chromatography (HPAC). The information obtained from this method can be useful in understanding how drugs interact with humic acid when they are circulated in environmental waters.

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[00:00:00.000]Hello, my name is Sazia
[00:00:02.530]and I am going to talk about creation
[00:00:04.620]of humic acid columns for analysis of drug binding
[00:00:08.680]and bioavailability in water.
[00:00:11.900]Drugs are often present in environmental water samples
[00:00:16.310]where they can undergo selective
[00:00:19.288]and reversible non-covalent interactions with humic acid,
[00:00:23.410]which is a large organic molecule present
[00:00:26.560]in environmental water.
[00:00:28.770]This results in the formation of a drug-humic acid complex.
[00:00:33.230]But there would always be a portion of the drug
[00:00:36.160]that does not bind to the humic acid.
[00:00:38.710]This is known as the free or unbound fraction of the drug.
[00:00:43.120]This free fraction of the drug crosses the cell membranes
[00:00:46.500]of aquatic animals, like fishes
[00:00:49.260]and imparts toxicity and causes harm
[00:00:52.920]or contamination in environmental waters and ecosystems.
[00:00:58.820]So there is an urgent need
[00:01:00.710]for the development of analytical tools
[00:01:03.505]that can characterize the interactions
[00:01:06.500]of drugs with humic acid
[00:01:08.790]so that we can get information about the bioavailability
[00:01:12.590]of drugs in environmental waters.
[00:01:15.500]So in this project,
[00:01:18.670]we developed a high-performance
[00:01:20.880]affinity chromatography technique
[00:01:23.670]in which we characterized the binding of four different drugs
[00:01:27.878]with a commercial preparation of humic acid
[00:01:31.260]by employing experimental conditions
[00:01:33.920]that replicated the conditions observed
[00:01:37.190]in environmental water samples
[00:01:39.663]as closely as possible.
[00:01:43.620]So what is humic acid?
[00:01:45.550]Humic acid is a large organic polymer,
[00:01:48.500]which is produced by the breakdown of plants
[00:01:51.370]and is present in environmental water samples,
[00:01:57.160]as well as soils and sediments.
[00:01:59.540]It has got a variety of functional groups
[00:02:02.440]in its structure.
[00:02:04.080]These includes the carboxyl group, phenols, and quinones.
[00:02:08.600]Along with these functional groups,
[00:02:10.220]it also has sugars and peptides in its structure.
[00:02:14.070]Through these functional groups,
[00:02:16.230]it can form non-covalent interactions with drugs.
[00:02:21.350]These non-covalent interactions
[00:02:22.936]include electrostatic interactions,
[00:02:25.610]hydrogen bonding and hydrophobic interactions.
[00:02:28.960]This binding ability of drugs
[00:02:31.472]with humic acid makes humic acid an important solubilizing
[00:02:36.620]and carrier agent for drugs
[00:02:39.090]in the environmental water samples.
[00:02:43.280]Since we are developing
[00:02:45.220]a high-performance affinity chromatographic method,
[00:02:48.260]we first prepared the stationary phase.
[00:02:51.220]So at first, we modified the silica support
[00:02:56.310]by treating it with oxalic dihydrazide.
[00:02:59.900]This led to the formation
[00:03:00.850]of a hydrazide-activated silica support.
[00:03:04.840]As we can see, we have hydrazide groups
[00:03:07.160]on the surface of the silica.
[00:03:09.740]Then we mixed the slurry of the hydrazide-activated silica
[00:03:14.210]with humic acid and oxidized glycogen.
[00:03:17.240]Now these hydrazide groups on the surface
[00:03:19.590]of the silica support do not form any direct bond
[00:03:23.420]with humic acid.
[00:03:24.890]Rather, they form a stable covalent bond
[00:03:28.300]with the oxidized glycogen,
[00:03:30.083]resulting in the formation of a cage-like structure.
[00:03:33.860]This cage-like structure entraps the humic acid
[00:03:37.110]in a fully soluble and active form
[00:03:39.940]within the pores or near the edges of the pores
[00:03:43.350]of the support.
[00:03:44.830]Once we have prepared
[00:03:46.140]the humic acid-entrapped silica support,
[00:03:48.778]we packed it
[00:03:49.820]into a stainless steel liquid chromatographic column.
[00:03:53.800]Then we introduced a small volume of the drug
[00:03:56.920]in presence of a pH 7.4 potassium phosphate buffer
[00:04:01.570]that also acted as a mobile phase in our study
[00:04:05.820]at a temperature of 25 degrees Celsius into our column.
[00:04:10.520]These conditions were selected
[00:04:12.274]to represent the environmental water system
[00:04:16.400]as closely as possible.
[00:04:18.450]As the drug passed through the column,
[00:04:21.190]it underwent selective
[00:04:22.960]and reversible non-covalent interactions
[00:04:25.740]with the entrapped humic acid in the support.
[00:04:28.957]These type of interactions are similar
[00:04:33.174]to the interactions we usually see
[00:04:36.090]between drugs and humic acid in natural
[00:04:39.640]or environmental water samples.
[00:04:42.750]Based on these selective interactions,
[00:04:45.570]we obtained our chromatographic response.
[00:04:48.920]Based on our chromatographic response,
[00:04:51.400]we obtain the retention time of the drug.
[00:04:54.060]This retention time of the drug allowed us
[00:04:57.330]to obtain or estimate the association equilibrium constant,
[00:05:00.920]which is actually the binding strength
[00:05:02.900]of the drug in presence of humic acid.
[00:05:07.310]In this project, we tested four drugs.
[00:05:09.797]The first drug was Carbamazepine,
[00:05:12.350]which is an anti-convulsant used to treat nerve pain
[00:05:16.171]and bipolar disorders.
[00:05:19.350]It can undergo hydrophobic interactions with humic acid.
[00:05:23.530]The next drug is the antibiotic Tetracycline,
[00:05:26.670]which can undergo electrostatic
[00:05:28.664]and hydrogen bonding with humic acid.
[00:05:33.160]It is usually used to treat skin infections.
[00:05:36.240]The next two drugs, Ciprofloxacin and Norfloxacin,
[00:05:40.030]belong to a class of fluoroquinolone antibiotics,
[00:05:44.340]which are used to treat serious infections,
[00:05:46.570]like urine infections and bladder infections.
[00:05:49.510]They usually associate with humic acid
[00:05:52.330]through hydrogen bonding.
[00:05:54.990]From our chromatographic results,
[00:05:56.970]we can see that each of the drug
[00:05:58.870]has a different binding strength in presence of humic acid.
[00:06:02.860]Different binding strength will mean
[00:06:04.720]that they have different bioavailability
[00:06:07.260]in the environmental water samples.
[00:06:09.860]Among the four drugs tested,
[00:06:11.790]the Norfloxacin exhibited the highest binding strength.
[00:06:15.130]The highest binding strength means
[00:06:17.000]that it had a lower bioavailable fraction.
[00:06:20.250]On the other hand, Tetracycline represented
[00:06:23.020]or showed the lowest binding strength,
[00:06:26.780]which means a higher fraction of the Tetracycline
[00:06:29.930]is present in environmental water in free form
[00:06:33.200]and that free form of the drug
[00:06:35.150]is going to impart toxicity to our aquatic ecosystems.
[00:06:40.030]So in summary, we developed
[00:06:42.110]a new high-performance affinity chromatographic method
[00:06:45.250]where we entrapped humic acid on a hydrazide activated silica support.
[00:06:49.210]These allowed us
[00:06:50.130]to characterize the drug-humic acid interactions.
[00:06:54.550]Based on our results, we saw that each
[00:06:57.470]of the drugs exhibited different binding strengths,
[00:07:00.710]which means that they have different bioavailability
[00:07:03.338]in water samples.
[00:07:05.900]That means they impart different amount of toxicity
[00:07:10.480]to the aquatic ecosystems.
[00:07:13.030]So in the future,
[00:07:15.010]we would collect environmental water samples,
[00:07:17.640]such as surface water and groundwater
[00:07:20.140]and then spike them with known concentrations
[00:07:23.180]of the drugs that we tested.
[00:07:25.640]And then we would inject those drugs
[00:07:28.680]into the column we made using the humic acid
[00:07:31.660]in this project.
[00:07:32.890]This would actually help us to quantify
[00:07:35.500]or characterize the binding strength
[00:07:37.730]of the drug in presence of humic acid
[00:07:39.940]in real water samples
[00:07:41.410]and give a better idea about the bioavailability
[00:07:44.377]of the drugs in environmental water samples.
[00:07:47.940]I would like to thank my research adviser,
[00:07:50.550]Dr. Hage and Dr. Snow for their help
[00:07:53.840]in designing this project
[00:07:55.550]and carrying out these experiments.
[00:07:57.880]This project has been kindly funded
[00:08:00.520]by the UNL Research Council
[00:08:02.450]under a Seed Grant.
[00:08:04.024]Thank you so much for listening to my talk.

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Creation of Humic Acid Columns for Analysis of Drug Binding and Bioavailability in Water

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