JPH03108662A - Liquid chromatography by packing and eluent containing surfactant - Google Patents

Abstract

PURPOSE: To achieve HPLC by injecting a sample containing a protein constituent directly into a reversed-phase high-performance liquid chromatography(HPLC) column without requiring the pretreatment process of the sample. CONSTITUTION: A sample is analyzed in an atmosphere where no surface-active agent micell exists since a surface-active agent in chromatography eluate exists at a concentration that is less than a critical micell concentration. Then, before the sample is introduced into a granular packing in a chromatography column for contacting, the sample is brought into contact with a solution for containing the same surface-active agent used for forming the eluate until the packing is saturated by the surface-active agent. After the packing is saturated by the surface-active agent, the packing can be brought into contact with the sample that contains a protein constituent without adsorbing a protein constituent or protein on the packing to the extent that the column performance cannot be lost. Then, the chromatography analysis of the sample is performed by the same eluate-surface-active agent composition as the one used for absorbing the surface-active agent in the packing.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method of performing chromatography for analyzing samples containing protein components.

[Prior art and problems to be solved by the invention] At present, reverse phase high performance liquid chromatography (HPLC)
) is difficult to perform in a short time on large quantities of samples containing protein components, such as biological samples. The main problem encountered with samples containing protein components is that proteins or denatured proteins are adsorbed onto the surface of the porous particles used in the PLC process.HPLC columns can become contaminated in a short period of time. It becomes a useless situation.

For this reason, samples are currently pretreated, for example by sedimentation or ultrafiltration.

Both pretreatments are undesirable as they are time consuming and labor intensive. Additionally, protein or denatured protein products are
There was a desire to use a small guard column in front of the HPLC column that would function to adsorb those proteins before they enter the LC column. Guard columns are cumbersome and expensive because they must be periodically discarded and replaced. Another method is to use internal surface reversal transport (ISRP) supports. The hydrophobic peptide partitioning layer only binds to the inner surface of the porous silica particles, while the outer surface is water permeable and non-adsorbent to proteins through the glycerylbrovir bonding layer. It is. Since the pores of silicate particles are sufficiently large,
-S Small molecule analytes penetrate the particles and undergo chromatographic resolution, but exclude globular proteins. This method requires that 15RP columns are expensive and their special phase 1) has poor retention and 2) is more unfamiliar to those operating the chromatography than normal reverse feed chromatography packing. Therefore, it is undesirable.

Recently, micellar chromatography has been used for direct injection analysis. This method is limited in scope and uses conventional reversed-phase chromagraphic packing, but uses an eluent containing a concentration of surfactant above the "critical micelle concentration" or CMC. The eluent generally contains little or no organic solvent to avoid inhibiting the formation of micelles. In such micellar eluents, the micellar aggregates in the eluent solubilize serum proteins and other components of the biological fluid and do not precipitate or adsorb these components onto the column packing and may be present in the sample. Allows reagents to be passed through the chromatography system without interfering with their analysis. However, some undesirable results have been reported regarding the use of micelles/8 female agents for direct injection analysis of reagents from biological fluids. For example, it has been reported that the elution time of a particular reagent decreases steadily over the course of a series of analyses.
In some cases, this is undesirable for routine analysis because it complicates the identification of chromatographic peaks based on elution time.

Therefore, there is a need to provide a method for performing HPLC by directly injecting a sample containing protein components into an HPLC column without the need for sample pretreatment steps. Additionally, there is a need to provide a means to avoid drift in the elution profile to enable use of the column for extended periods of time.

[Means for Solving the Problems] The present invention provides a method for applying a surfactant to a chromatographic packing using a solution containing a surfactant at a concentration below the critical micelle concentration (CMC). Based on the discovery that the resulting modified packings can be used in HPLC on protein-containing samples, thereby causing little or no adsorption of protein components onto the packings. The term "protein component" as used herein refers to proteins and/or denatured protein products. Eliminates the need to use concentrations of surfactant at or above the chromatography column by pumping the surfactant solution through the chromatography column and monitoring the eluate therefrom until it has a constant surfactant concentration; It has been demonstrated that the packing is saturated with surfactants, so that the chromatography column
Suitable for receiving protein-containing samples without adsorption of proteins onto the column packing; Inject an eluent-surfactant solution similar to that used to adsorb the active agent.

In the chromatographic analysis method of the present invention, a sample is analyzed in the absence of surfactant micelles. This is while the surfactant in the chromatographic eluent is present at a concentration below the critical micelle concentration. Prior to introducing and contacting the sample with the particulate packing in the chromatography column, the packing is injected into a solution containing the same surfactant used to form the eluent, while the packing is injected into a solution containing the same surfactant used to form the eluent. contact until saturated. After the packing is saturated with surfactant, the packing can be contacted with a sample containing protein components without adsorbing the protein components or proteins onto the packing to the extent that column performance is compromised. Chromatographic analysis of the sample
Run with the same eluent-surfactant composition used to absorb the surfactant into the packing. To ensure that the surfactant coats the column packing, it is necessary to first monitor the adsorption process that occurs when the column is exposed to the surfactant-containing eluent. This can be done, for example, by recording the response of a differential refractometer. Once the surfactant is adsorbed onto the column packing, a baseline level is observed. After a sudden rise or fall in the baseline, the signal stabilizes at a new level, indicating that the surfactant is no longer adsorbed. Following the refractometer, knowledge of the surfactant in the eluent, i.e. its concentration, allows calculation of the amount of surfactant adsorbed. In one embodiment of the invention, an eluent modifier can be added to the surfactant solution that increases the tendency of the surfactant to be absorbed onto the column.A representative suitable eluent modifier is phosphate. The surfactants used in the method of the present invention are species that exhibit both hydrophobic and hydrophilic properties. They consist of two parts with clearly different properties.

There is a polar "head group" which can be cationic, anionic, neutral or zwitterionic, and a hydrocarbon tail of varying chain length. This dual nature of nature gives surfactants their unique properties, among which they tend to absorb at the interface, where both hydrophobic and hydrophilic properties are most satisfied.

Micelleization, which should be avoided in the method of the invention, is the result of opposing forces, and the driving force is the hydrophobic effect. Beyond the CMC, which exists for all surfactants, surfactant molecules self-assemble and
With the hydrocarbon tails oriented toward the center of the assembly and the polar head groups facing outward, this self-assembly acts to sufficiently eliminate the hydrocarbon-water interface (so that micellization is not fully achieved). However, the mutual repulsion of the head groups is the dominant force controlling the size and shape of the micelles.

For these reasons, the structure of micelles depends somewhat on the properties of the solution, such as ionic strength, the presence of small amounts of organic solvent, and the chemical properties of the solute.

Micelles can be small spheres or plates, oblate or prolate ellipsoids, or long cylinders. Their dimensions generally range from 3 to 6 nm in diameter, which allows their macroscopic solution properties to approach those of truly homogeneous solutions. They cannot be filtered using conventional methods, and they do not produce observable light dispersion errors in visible-ultraviolet absorption spectroscopy; micellization does not occur at precise predetermined concentrations, but is rather narrow; It is a process that occurs over a range of 1 degree.

The reported CMC values are based on measuring the change in slope of the appropriate physical property that distinguishes between micelles and free amphoteric medium when plotted against total concentration. For this reason, various methods of CMC quantification yield slightly different values. Above the CMC, there is an approximately constant amount of free surfactant in solution, the concentration of which is equal to the CMC.

The surfactant used depends on the column packing used, the nature of the solute being analyzed and the nature of the eluent used. The procedure for quantifying useful surfactants for a given combination of analytical conditions is listed below:・Chromatography Department (Massachusetts,
A surfactant-containing knee bondapak (uBondapak Cl8), commercially available from Milford, is prepared.

2. Inject 50 μm of serum into a chromatography system incorporating the column and eluent described above.

3. Observe the peak area of the first eluting serum peak in the resulting chromatogram.

4. Ni Bondervac C18 containing surfactant, commercially available from Millipore's Waters Chromatography Division (Milford, Mass.);
Manufactured with a new surfactant (CMC unvortexed, 0 concentration).

5. Repeat steps 2 and 3 above. The peak areas should be almost the same. If the peak area for the new surfactant is substantially less, the surfactant is either not suitable or its concentration is too low.

6. If a packing other than Knee Bonderback C+a is used, steps 4 and 5 should be repeated with the new packing.

1. Make a surfactant-containing packing with any packing and any surfactant that results in a concentration below the CMC in the eluent.

2. Temporarily remove the chromatography column from the liquid chromatography system.

3. Inject 50 μm of serum.

4. The resulting peak was captured and the sample was subjected to Bradford (3radford) analysis for protein analysis or another suitable analysis.

5. Reinstall the column into the system. Steps 3-4 were repeated and compared with the results of the Lowry analysis. They matched perfectly. If the results obtained with the column are substantially less than the results obtained without the column, the selected combination of surfactant, column packing, and eluent is not suitable.

Suitable surfactants are anionic, nonionic and amphoteric surfactants. Representative suitable anionic surfactants are sodium dodecyl sulfate (SDS) in water, water/methanol, or buffered water/methanol, octane sulfonic acid in water. Representative suitable nonionic surfactants include polyoxyethylene 23 lauryl ether in water, sodium desoxycholate in water, water/methanol or buffered water/
Representative suitable zwitterionic surfactants include 3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate (CHAP) in water and the like.
S), water/methanol or buffered water/methanol, etc.

Suitable particulate column packings include inorganic materials with 18-aliphatic groups. Such packing materials are available under the trademark Bondapak from Millipore's Waters Chromatography Division, Milford, Mass.
f:+s/Porasil B and nee Bonderback/-Coras H and Bondervac C18. Other suitable column packing materials include hlicroBondapa phenyl
K Phenyl), Tsubabak (NovaPak) C
18, Tubavac phenyl or Tubavac cyanide (C
yane).

The method of the invention is particularly useful for chromatographic analysis of biological fluids such as serum, plasma or urine samples that are injected directly into a chromatography column. Biological samples can be analyzed for drugs or drug metabolites, for example.

The present invention provides significant time savings since sample contamination is minimal. Serum samples are briefly filtered and injected into a liquid chromatography column. If necessary,
On the condition that a column filter is installed in liquid chromatography? The total throughput of zero analysis, which can even omit filtration, is 20% lower than that of a column that is not saturated with surfactant.
It can be increased by a factor of ~50. Furthermore, a wide range of organic modifier compositions can be utilized to provide a wide range of flexibility in implementing existing liquid chromatography analyses. Adjust retention to accommodate a wide variety of solutes. Furthermore, the method of the invention allows for the analysis of all protein-bound reagents. Conversely, analysis of protein binding reagents currently requires cumbersome sample preparation prior to analysis.

Furthermore, since the protein was eluted with a significant loss, there was no reduction in column lifetime due to protein adsorption or precipitation.

Examples illustrating the present invention are shown below, but they do not limit the present invention in any way.

EXAMPLE This example illustrates the analysis of solutions containing serum proteins by the method of the invention.

In this example, a model 510 pump and a W I
A liquid chromatograph equipped with an SP-710B or 712 syringe (all commercially available from Millipore's Waters Chromatography Division, Milford, Mass.) was used. Column eluent was monitored by a 254 nm UV detector. The liquid flow rate through the column was approximately lag/min. 7.5 cm internal diameter x 3.9 m commercially available from Millipore's Waters Chromatography Division, Milford, Mass., under the trademark Tubavac Phenyl.
A pre-packed column of m was used. Tubavac Phenyl is a 35% (w/w) solution of 1 mmol sodium dodecyl sulfate (SDS) with a monolayer of dimethyl-propylphenylsilane chemically bonded to spherical silica beads with an average size of 4 microns. ) An aqueous methanol solution was pumped through the column and the column eluent was monitored with a differential refractometer.

After about an hour, a sudden rise in the baseline of the previous level was observed, followed by a steady-state of the signal at a new, higher level.

The signal was detected when the column was injected with SDS at a concentration of approximately 2 mg per column.
indicates saturation. There were no micelles in this eluent.

The sample solution was prepared with carbamazepine and carbamazepine epoxide at known concentrations of 22 μg/ld and 5 μg/ld, respectively.
It consisted of 50 mg/- of bovine serum albumin. The results are shown in Figure 1, which shows that 500 analyzes were performed for each sample component with good repeatable retention times.

To confirm whether the protein was adsorbed onto the column, BioRad for protein weight recovery was used.
d) (Bradford) analysis was used to quantitatively confirm that bovine serum alpvinmin elutes early in the chromatography run. The analysis was performed as follows.

Bio-Rad's commercial operation of the Bradford protein assay is based on the color change of Commassie Brilliant Blue G-250 dye in response to varying concentrations of protein (a dye-binding assay). Bradford's analysis was performed using a protein standard sample (in this study, bovine serum albumin).
BSA)) Response to concentration (absorbance at 595 nm)
A standard curve needs to be created. The standard sample protein is 15% (w/w) methanol in water with a 1 mmolar concentration of SDS added to the chromatographic eluent to be tested at various concentrations. The response for each standard protein solution was measured for the amount of solution needed to fill the cell used in the UV-Vis spectrometer.The reference cell of the spectrometer was filled with protein-free chromatography eluent. Thus, response values were corrected for background absorbance.

After generating the standard curve, the protein concentration for a solution containing an unknown concentration of BSA was determined by measuring the response and reading the corresponding concentration from the standard curve. If the response is higher than the range contained within the standard curve, the sample must be diluted by a known factor before being measured. Later adjust the calculated concentration of protein using the dilution factor.

In the context of the present invention, using the Bradford analysis, when using specific column packing and chromatographic eluents, 50 mg/- of a 50 mg/- solution of BSA
li? that an injection of 0-100 μl yields an eluent of 00% B5Al? ! I approve. To accomplish this, the amount of BSA eluted is measured with and without the column. If the results are the same, the BSA eluent is 100%. If less BSA elutes than without the column, the chromatographic eluent being tested is not suitable for use according to the method of the invention.

The above analysis shows that 100% of the injection volume is removed from the column by the surfactant-containing eluent; in this manner, more than 500 analyzes were performed without abnormally changing system pressure and chromatographic performance. executed continuously. This proves that the protein is not precipitated and adsorbed onto the packing.

This example describes the analysis of theophylline, theobromine, and caffeine from solutions containing proteins by the method of the present invention.

Modular liquid chromatography equipped with a Model 510 pump and WISP712 injector (all commercially available from Millipore's Waters Chromatography Division, Milford, Mass.)
was used in this example. The column eluate was monitored by a UV detector at 254 nm. The liquid flow rate through the column was approximately lag/min. A 7.5 cm by 3.9 mm internal diameter prepacked column sold under the trademark Tubavac Phenyl by Millipore's Waters Chromatography Division (Milford, Mass.) was used. Camellia phenyl CI
The IB packing has a monolayer of dimethylpropylphenylsilane chemically bonded to spherical beads with an average size of 4 microns. A solution of 1 mmol of sodium dodecyl sulfate (SDS) in 15% (w/w) methanol in water was pumped through the column and the column eluent was monitored by a differential refractive index system. After 1 hour, a sudden rise in the previous baseline level was observed, followed by stabilization of the signal at a new, higher level. The signal is about 7 columns per column.
．． It shows that the concentration of 5B was saturated at 5DSD. No micelles were present in this eluent.

The protein-containing solution consisted of 50 mg/- bovine serum albumin with known concentrations of theophylline and theobromine. The results are shown in Figure 2. The close match between the 200 and 520 chromatograms indicates that good chromatographic performance was maintained for at least 520 analyses.

This example illustrates the analysis of solutes from serum by the method of the invention.

Modular liquid chromatography equipped with a Model 510 pump and WISP712 injector (all commercially available from Millipore's Waters Chromatography Division, Milford, Mass.)
was used in this example. Column eluent was monitored by a 214 nm UV detector. The flow rate of liquid through the column was approximately 2aj/min. A 15 cm x 3.9 mm internal diameter prepacked column commercially available from Waters Associates under the trademark Nybonderback Phenyl was used. The knee bonderback phenyl packing has a monolayer of dimethylpropylphenylsilane chemically bonded to spherical silica beads with an average size of 10 microns. A 25% (vol/vol) aqueous solution of 5 mmol of sodium dodecyl sulfate (SDS) in methanol was pumped through the column and the column eluent was monitored by a differential refractive index system. After about 15 minutes, a sudden rise in the previous baseline level was observed, followed by a stabilization of the signal at a new, higher level. No. 3 indicates that the column was saturated with SO3 at approximately 35 mg per column. No micelles were present in this temperate solution. serum.

The results obtained are shown in FIG. The figure shows that the retention time for the analyte remained stable after 500 injections. Peak areas were also stable and no pressure problems occurred provided normal maintenance procedures were followed.

Chromatography was carried out 200 times and 52 times.
This is a chromatogram obtained when the experiment was carried out 0 times.

FIG. 3 is a graph showing the results of chromatography of adult cow serum to which carbamate zevin evoside was added according to the method of the present invention, in terms of retention time versus number of injections.

4. Figure 1 shows the method of the present invention for a sample composed of 50 mg/- of bovine serum albumin containing carbamazepine and carbamazepine evolocyto at known concentrations of 22 μg/- and 5 μg/al, respectively. It is a graph showing the change in retention time with respect to the number of injections when analysis was repeated 500 times.

Figure 2 shows the retention times of the method of the invention Figure 3 for bovine serum albumin samples containing theophylline and theobromine.

Claims (10)

[Claims] (1) A liquid chromatography method for analyzing a composition containing a protein component for solutes dissolved in the composition, the composition comprising: passing the composition through a column having a packing material in an eluent containing a surfactant; packing of the protein component, wherein the packing material is coated with a substantially maximum amount of the surfactant adsorbed on the packing material, thereby impeding the passage of the solute through the column; The above method for preventing adsorption to. (2) The method according to claim 1, wherein the composition containing the protein component is serum. (3) Claim 1, wherein the composition containing the protein component is urine.
the method of. (4) The method according to claim 1, wherein the composition containing the protein component is plasma. (5) The method of claim 1, wherein the surfactant is an anionic surfactant. (6) The method of claim 1, wherein the surfactant is a nonionic surfactant. (7) Claim 1, wherein the surfactant is an amphoteric surfactant.
the method of. (8) The method of claim 4, wherein the anionic surfactant is sodium dodecyl sulfate. (9) The nonionic surfactant is polyoxyethylene (
23) The method of claim 6, wherein the lauryl ether is lauryl ether. (10) The method of claim 7, wherein the amphoteric surfactant is 3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate.