IAM Care And Use

The Regis IAM.PC.DD Drug-Discovery
HPLC Column
It's Nature, Care, and Use

1. Introduction
2. The Nature of the IAM.PC.DD 2 Drug-Discovery Column
3. Attaching the IAM.PC.DD 2 Column to the Chromatograph
4. Mobile Phase Make-Up
5. Conditioning the IAM.PC.DD 2 Drug-Discovery Column
6. Calculating Capacity Factors (k'IAM)
7. Column Cleaning and Storage
8. References
9. Technical Assistance

I. Introduction

Noninvasive methods of drug delivery are a highly desired property of new drug candidates. Since, oral drug absorption experiments in animals are both experimentally difficult and expensive, a number of in vitro models for predicting oral drug absorption have been developed.

Chromatographic models to predict drug absorption are much easier to use than other partitioning, cell culture, and animal models. Among chromatography columns, the IAM.PC.DD 2 Drug-Discovery column is significantly effective in modeling the hydrophobic and hydrophilic contribution to a drug s partitioning.

II. Nature of the IAM.PC.DD 2 Drug-Discovery Column

Immobilized Artificial Membrane (IAM) chromatography columns mimic the lipid environment found in cell membranes1,2. Phosphatidylcholine (PC) is the major phospholipid found in cell membranes and IAM columns prepared from PC analogs are models of cell membranes. Drug discovery frequently requires understanding drug membrane interactions and the new IAM.PC.DD 2 column can be used for elucidating drug-membrane interactions. It models the hydrophobic and hydrophilic contribution of a drug s partitioning.

For small molecules, the membrane partition coefficient, Km, is the rate limiting step for drug absorption. IAMs can be used to measure the partitioning of drugs to fluid membranes. Similar to fluid membranes, a drug will exhibit an equilibrium constant, K IAM, to the immobilized membrane. Since the capacity factor, k'IAM, is linearly related to K IAM, measuring k'IAM is effectively the same as measuring K IAM for the purpose of drug discovery.

The IAM.PC.DD 2 column has been specially designed for the precise and reproducible measurements needed for large scale drug absorption screening.

The structure of the IAM.PC.DD 2 packing is shown.

Three IAM.PC surfaces were studied to determine membrane partition coefficients2. Each IAM phase gave virtually identical results for predicting drug membrane partitioning and drug membrane transport. Complementing our current IAM.PC.DD Drug-Discovery column, Regis now offers the IAM.PC.DD 2 column. Possessing similar characteristics to the IAM.PC.DD packing, the ester bonding of the IAM.PC.DD 2 packing offers more hydrophobicity, thus giving longer retentions to compounds not well retained on the IAM.PC.DD packing. Retentions are typically double on the IAM.PC.DD 2 packing but still exhibit excellent correlation for groups of compounds. (See Table 1).

Table 1. Correlating Drug Partitioning into IAM With Rat Intestinal Drug Absorption

Sample	% Absorption of Inverted Rat Intestine	(k') IAM.PC.DD	(k') IAM.PC.DD2
m-nitroaniline	77	3.875	10.838
p-nitroaniline	68	8.576	16.086
salicylic acid	60	1.936	6.963
p-toluidine	59	1.554	4.546
aniline	54	0.811	2.069
m-nitrobenzoic acid	53	1.213	4.403
phenol	51	2.965	6.544
benzoic acid	51	0.531	2.088
acetanilide	42	1.797	5.096
antipyrine	32	0.685	3.350
theophylline	29	0.583	1.478
acetylsalicylic acid	20	0.197	0.931
r (correlation factor)		0.8088	0.8025

Chromatographic conditions for Table 1: Columns: 10 cm x 4.6 mm Mobile Phase: 0.01M DPBS, pH=5.4 Flow rate: 1.0 mL/min

Another distinct advantage of the IAM.PC.DD 2 packing is its ability to tolerate mobile phases of pH 7.0 or higher. The IAM.PC.DD 2, at pH's greater than 7.0, is more stable thus giving it longer column life.

The ligand is bonded on 12 µm, 300 Å, spherical aminopropyl silica and endcapped with C10 and C3 amides. It is a chromatographically efficient packing.

III. Attaching the IAM.PC.DD 2 Drug-Discovery Column to the Chromatograph

Column connections are an integral part of the chromatographic process. If ferrules are overtightened, not set properly, or are not specific for the fitting, leakage can occur. Nuts and ferrules are provided with each column and should be used for all column connections.

Set the ferrules for column installation to the HPLC system as follows:

1. Place the male nut and ferrule, in order, onto a 1/16" o.d. piece of tubing. Be certain that the wider end of the ferrule is against the nut.
2. Press tubing firmly into the column endfitting. Slide the nut and ferrule forward, engage the threads, and fingertighten the nut.
3. While continuing to press the tube firmly into the endfitting, use a 1/4" wrench to tighten the nut 90 degrees past fingertightness.
4. Repeat this coupling procedure for the other end of the column.

IV. Mobile Phase Make-Up

All buffers should be made with HPLC grade water and filtered prior to use. The pH of the buffers should be adjusted to match the closest physiological pH of the samples. The pH range of the IAM.PC.DD 2 columns is between 2.5 and 7.5.

Note: Guard columns should be used at all times

V. Conditioning the IAM.PC.DD 2 Drug-Discovery Column

To condition the column, pass approximately 10 to 15 column volumes (10 to 15 mL for the 3 cm column; 20 to 30 mL for the 10 cm column) of mobile phase through the column or until a stable base line is achieved. Make several repetitive injections of one compound to insure identical retention times of that compound. Once the retention times are identical, the system is fully equilibrated and ready for use.

VI. Calculating Capacity Factor

K IAM is obtained by measuring the IAM capacity factor, k'IAM. The capacity factor is calculated from the retention time t r and the column void volume time t0 from the following equation:

k'IAM  =	tr - t0
	t0

The procedure for determining the capacity factors for the test compounds is as follows:

1. Determining the void volume time t0

When the system is fully equilibrated, dissolve approximately 50 g of citric acid in one mL of mobile phase. With the mobile phase flow rate set at 1 mL/min, inject 10 L of this solution into the chromatograph. The retention time (time from injection to the peak apex) is the void volume time of the system (t0). Calculate the retention time of individual compounds in a similar manner.

Using these retention values (tr) and the t0 for the system, calculate the capacity factor (k'IAM) for each of the compounds using the above formula.

Correlation coefficients are determined by plotting (log % Absorbance) Vs (log k'IAM).

Figure 1 illustrates a typical plot determined using data from Table 1.

VII. Column Cleaning and Storage

To clean, reverse the column, then wash with 10 to 15 column volumes of water, followed by 10 to 15 column volumes of acetonitrile. When the column is to be taken out of service for an extended period, acetonitrile should be used as the storage solvent.

The column is to be kept wet during storage. Each column is shipped with two removable end plugs, to prevent the drying of the column bed. Save these plugs and re-install them whenever the column is removed from the HPLC system.

VIII. References

1. Pidgeon, C., et al., J. Med. Chem. 1995, 38, 590-594.
2. Ong, S.: Liu, H.: Qiu, X.: Bhat, G.: Pidgeon, C. Anal. Chem. 1995, 67, 755-762.
3. Schanker, L. S.: Tocco, D. J.: Brodie, B. B.: Hogben, C. A. M. J. Pharmacol. Exp. Ther. 1958, 123, 81-88.

IX. Technical Assistance

If you have any questions concerning the performance or application of this column, please contact Regis Chromatography Department at (800) 323-8144, (847) 967-6000, or FAX (847) 967-5876. or email Ted Szczerba.

Regis has made every effort to present accurate information in this booklet. However, users should use their own judgement to determine the suitability of any of the products, data, or procedures presented.

Note: These columns are not intended for use as medical devices, nor for resale for such uses.