GB2042557A

GB2042557A - Filler composition for use in liquid chromatography

Info

Publication number: GB2042557A
Application number: GB8003946A
Authority: GB
Original assignee: Kureha Corp
Current assignee: Kureha Corp
Priority date: 1979-02-07
Filing date: 1980-02-06
Publication date: 1980-09-24
Also published as: FR2448718A1; DE3004356A1; GB2042557B; CA1145737A; JPS55106357A; FR2448718B1; IT1150076B; JPS6331737B2; DE3004356C2; IT8019759A0

Abstract

A filler composition for use in a liquid chromatography column comprises beads of a hydroxymethylated copolymer of styrene and divinylbenzene having a serum protein adsorbed or adhering thereonto. Columns packed with such beads can be used in clinical examinations and enable high-speed chromatographic techniques to be employed such that separate peaks can be detected on a chromatogram within a short operation time and using only a small amount of specimen fluid.

Description

SPECIFICATION Filler composition for use in liquid chromatography The present invention relates to a filler composition for use in a liquid chromatography column, to a process for preparing such a filler composition, to a column packed with the filler composition and to a method of liquid chromatography using such a column.

It is extremely important for diagnosing and treating diseases to obtain information on the state of the disease by analysing the properties and the components of the patient's physiological fluids such as plasma, serum, cerebrospinal fluid and urine.

Hitherto, such information has been obtained by various chemical and biochemical analytical means.

However in many cases a method of obtaining exact information on the state of the disease is required.

Particularly in cases of diseases of the kidney and of the liver, because of their complicatedness, further information about them is continually being sought. Conventionally data concerning a variety of properties associated with the function of each organ is determined by analytical techniques. For example in the case of renal diseases, the levels of creatinine, uric acid and electrolytes in blood, protein and sugar in urine and the pH of urine are monitored. In the case of hepatic diseases, the activities of bioenzymes, for instance GOT, GPT, LDH and LAP, and the components of blood, for instance protein and bilirubin are analysed. This data plays an important role in establishing the diagnosis and the guiding principles for the treatment of a particular illness.

However, recently it has been found that there is an intimate relationship between uremictoxins and the state of renal disease. The abovementioned values, which are determined by conventional methods, are not able to confirm the presence of such toxins. Moreover, no simple method for detecting and confirming the presence of the toxins has yet been developed. Also, in the case of hepatic diseases, in spite of the suggestion of the presence of various abnormally metabolised substances and of etiopathogenic substances, there are no suitable methods for detecting such substances at present. In short, the present chemical and biochemical analytical methods, although useful, are not entirely satisfactory.

In consideration of this situation, the utilization of liquid chromatography, which is an analytical method based on different principles from those of chemical and biochemical analytical methods, has recently attracted attention. It is possible to determine thermally and chemically unstable substances by liquid chromatography without their denaturation. Several components can be determined from a relatively small amount of a specimen. Accordingly, in principle, the applicability of the liquid chromatography to medicine is large. However, in order to put liquid chromatography to practical use, there have been problem concerning the selection of the filler material for the chromatographic column, the establishment of conditions for separation and pre-treatments which may need to be adapted.

For instance, Chang eft at "Trans. Amer, Artif. Int.

Organs", Vol. XX, page 364 (1974) tried to detect uremic toxins appearing in the blood of a patient suffering from a renal disease by using liquid chromatography with columns filled with porous beads of cross-linked dextran. They found that in the serum of the patient suffering from the renal disease there are some substances which give specific peaks in the chromatogram obtained by their method of liquid chromatography which do not appear in chromatograms of serum obtained from healthy patients. They suggested that these peaks could include some harmful substances.

However, there are defects in the method of Chang etc!. The specific peaks obtained are extremely broad and the method necessitates a relatively large serum sample (2 to 3 ml). Moreover, it takes 4 to 7 hours to treat one sample. Accordingly the method is not satisfactory as a clinical method of examination.

Furst"Clinical Nephrology", Vol 5 (4), page 198 (1976) proposed a method of analysis of serum specimens by high-speed liquid chromatography.

However, although Fürst's method was successful in shortening the time period for analysis, many peaks overlap each other in the chart that is obtained. This makes separation and identification of each component difficult.

In addition, neither of the two methods have been tried with the intention of employing them in clinical examinations. That is, although the analysis of biospecimens by liquid chromatography has been tried and it has been shown that it is possible to detect some specific peaks in the chromatogram of a specimen from the patient suffering from a disease of the liver or kidney, the results that have been obtained do not convey any information on the relationship between the progress of the disease and the appearance of the peaks in the chromatogram.

Accordingly, neither the method of Chang northatof First has been put to practical use.

It has now been found by using a particular filling composition for a liquid chromatography column, it is possible to separate and detect the peaks in a chromatogram which provide information on the progress of the state of either liver or kidney diseases within a shorttime period employing only a small amount of a specimen.

Accordingly the present invention provides a filler composition for use in a liquid chromatography column comprising beads of a hydroxymethylated copolymer of styrene and divinylbenzene, which beads have a serum protein adsorbed or adhering thereonto.

The present invention also provides a process for preparing such a filler composition, which process comprises bringing beads of a hydroxymethylated copolymer of styrene and divinylbenzene into contact with a solution of a serum protein.

The invention further provides a method of liquid chromatography which method comprises passing a liquid through a column packed with the filler composition of the present invention. It is possible to use this technique for clinical examinations and highspeed liquid chromatographic methods can be emp loyed.

In the description which follows, reference will be made to the accompanying drawings in which: Figure 1 shows chromatograms of serum speci mensobtained by the high-speed liquid chromatographic method of Example 1. Chromatogram (a) is of normal serum, and chromatogram (b) is of the serum of a patient suffering from renal failure.

Figure 2 shows chromatograms of serum specimens obtained by the high-speed liquid chromatography method of Example 3. Chromatogram (a) is of normal serum, chromatogram (b) is of the serum of a patient suffering from a liver disorder, and chromatogram (c) is of the same patient after recovering from the liver disorder.

The filling composition for use in the column of the liquid chromatography according to the present invention comprises the bead-like substance consisting of a hydroxymethylated copolymer of styrene and divinylbenzene, to which serum protein has been adsorbed or is adhering.

The filling composition of the present invention is prepared as follows: (a) The preparation of the bead-like substance comprising a hydroxymethylated copolymer of styrene and divinylbenzene: For instance, a monomeric mixture of styrene and divinylbenzene is polymerized in suspension in a non-solvent, for instance, in water in the presence of a polymerization initiator to obtain a bead-like copolymer of styrene and divinylbenzene. By bringing the bead-like copolymer into reaction with formaldehyde, a hydroxymethylated copolymer of styrene and divinylbenzene is obtained. The com mercialized bead-like copolymer of styrene and divinylbenzene hitherto available for use in the column of the high-speed chromatography as the filler may be applied as well as in the following step.The particle size of the bead-like copolymer and its degree of hydroxymethylation are possibly selected respectively in accordance with the purpose of the liquid chromatography, however, the former is usually 5 to 50 micron in diameter and the latter is usually 0.05 to 0.5.

(b) Adsorption of adherence of serum protein onto the bead-like oxymethylated copolymer of styrene and divinylbenzene: A serum protein, for instance, albumin and grobulin, obtained from a broad range of mammals such as mankind, cattle, horse, dog or sheep is dissolved in a solvent mentioned as follows, and the solution is brought into contact with the above-mentioned bead-like hydroxymethylated copolymer under agitation and then the system is separated into the liquid and the solid to obtain a hydroxymethylated copolymer of styrene and benzene having a serum protein adsorbed or adhering thereonto. The above-mentioned contact process is executed by immersing the above-mentioned hydroxymethylated bead-like copolymer into the solution of serum protein or by passing the solution of serum protein through a column filled with the bead-like hydroxymethylated copolymer.In addition, adsorption or adhesion is automatically completed only by bring ing the above-mentioned two substances into contact with each other because of the adsorbing facility of the hydroxymethylated copolymer for the serum protein.

The above-mentioned solution of serum protein used for adsorption or adhesion is obtained by dissolving the protein into water, a buffer solution or a buffer solution containing a salt such as sodium perchlorate or an organic solvent such as methanol, ethanol, propanol and dioxan. In cases where the concentration of the serum protein in the abovementioned solution is high, the adsorption or adhesion of the serum protein to the hydroxymethylated copolymer is completed within a short time period, however, the state of the adsorption or adhesion tends to be non-uniform, while in cases where the above-mentioned concentration is low, it takes a long period of time for the completion of adsorption or adhesion. In such circumstances, the concentration of the serum protein in the above-mentioned solution is preferably 0.2 to 5% by weight.

As is mentioned above, after having the serum protein adsorbed or adhering onto the bead-like hydroxymethylated copolymer of styrene and divinylbenzene, the filling composition of the present invention is obtained by washing well the beadlike copolymer thus treated. In order to apply the filling composition into the liquid chromatography, the filling composition is filled into the column for use in the chromatographical analysis. However, since the interparticle coagulative tendency has been raised by the adsorption of the serum protein, the uniform filling of the filling composition into the column has come to be difficult. Care should be paid on this fact.In order to avoid the troubles on the filling of the column with the filling composition of the present invention, it is recommended that the hydroxymethylated copolymer not yet having adsorbed the serum protein is in advance filled into the column and after equipping the filled column onto the conventional apparatus of the liquid chromatography, the solution of the serum protein is passed through the column. According to the above-mentioned technique, the time required for the serum protein to be adsorbed orto adhere onto the bead-like hydroxymethylated copolymer becomes longer, while on the other hand there is a merit of simplification of the preparatory operation for the analysis.

On the treatment for adsorption or adhesion of the serum protein onto the bead-like hydroxymethylated copolymer, the temperature is kept at a degree at which denaturation of the serum protein does not occur, that is, 5 to 70"C, preferably at 20 to 400C.

The pH of the solution of the serum protein may be in the range in which the denaturation and coagulation of the serum protein do not occur, and is usually selected suitably from the range of 5 to 9, however, it is natural to avoid the isoelectric point of the serum protein. In addition, on the preparation of the solution of the serum protein, the use of a buffer solution having the same pH value as the pH value of the moving phase used for the liquid chromatography carried out by using the column filled with the filling composition of the present invention facilitates the maintenance of the steady state of the solution of the serum protein during the operation of the examination and is suitable for the purpose.

Accordingly, the adsorption or adhering of the serum protein onto the bead-like hydroxymethylated copolymer is preferably carried out under the same conditions as far as possible to those at the time of examination, in considering the stability of the filling composition of the present invention.

The amount of the serum protein adsorbed or adhering onto the bead-like hydroxymethylated copolymer of the present invention varies slightly corresponding to the kinds of the bead-like hydroxymethylated copolymer and those of the serum protein for use in the liquid chromatography, however, usually in the range of 0.1 to 1% by weight of dry matter, preferably 0.2 to 0.5% by weight of dry matter.

The column filled with the filling composition of the present invention is possibly used for examination of various physiological specimens after installing the column onto any commercial apparatus for liquid chromatography or onto any other apparatus having the same function as above, and the thus installed apparatus is able to separate and detect the components of the specimen as the peaks which have relationships to the progress of the morbid state. In addition, the physiological specimens mentioned in the present invention include the blood components such as serum and plasma, cerebrospinal fluid, lymphs, ascite, urine, etc.

In the next place, the method of examination utilizing the filling composition of the present invention, particularly the method of examination of physiological specimens by the high-speed liquid chromatography can be executed under the following conditions, however, the followings only illustrate the mode of its execution referring to examples not being limited in themselves.

As the moving phase for use in the column filled with the filling composition of the present invention, water, a buffer solution or the buffer solution containing some salt component such as sodium perchlorate or an organic solvent such as methanol, ethanol, isopropyl alcohol, dioxan, etc. is preferable.

Particularly preferable material as the moving phase is a phosphate buffer, and by using the phosphate buffer as the moving phase extremely finely separated peaks are available in the chromatogram.

In addition, since the filling composition of the present invention contains the protein and accordingly there is a probability of suffering from undesirable transformations such as the degradation by microorganisms during the long time period of using the column filled with the filling composition of the present invention, it is preferable in those cases to add a minute amount of an anti-microbial agent such as sodium azide, etc. to the moving phase.

The temperature at which the examination is carried out by using the liquid chromatography utilizing the filling composition of the present invention is 20 to 40"C. The amount of the specimen required for carrying out the analysis by the abovementioned apparatus may be 1 to 15 microliter.

The liquid chromatography according to the present invention is provided with a detector comprising an ultra-violet spectrophotometer and an ordinary spectrophotometer, however, other than those mentioned, a differential refractometer, a fluorescence spectrophotometer, an infrared spectrophotometer, a radiation detector, a polarograph or a conductometer may be optionally utilized after careful selection.

In addition, in order to quantify the obtained data, the peak area on the chromatogram is possibly quantified by connecting a data-treating machine to the above-mentioned detector.

The liquid chromatography utilizing the filling composition of the present invention is not only applicable to the clinic examination but also, for instance, to separately collecting fractions afterfil- ling into a larger column.

The present invention is explained as follows by referring to the Examples, however, the scope of the present invention is not to be limited within Examples.

Example 1: After filling a hydroxymethylated bead-like (10 to 15 microns in diameter) copolymer of styrene and divinylbenzene prepared by a well known process into a stainless-steel column of4 mm in diameter and 50 cm in length by an ordinary technique, the column was installed onto a high-speed liquid chromatagraph provided with an ultraviolet detector.

Into the thus installed column, a phosphate buffer of pH of 7.4 was introduced art a rate of 1.2 ml/min and after confirming that the indication of the recorder and of the integrating planimeter became stabilized, an aqueous 10% solution of human serum albumin was introduced into the column from the specimen-inlet. After repeating the introduction of the solution of albumin to obtain the fixed height of the indication on the recorder or the fixed value of the indication of area on the integrating planimeter, the treatment of the bead-like filler with the abovementioned solution of albumin was completed.

Then, using the above-mentioned high-speed liquid chromatograph provided with the thus prepared column in which the bead-like filling had adsorbed or adhered to the human serum albumin completely, the examination of the serum of a normal person and the serum of a patient of chronic renal failure under dialytic treatment was carried out.

As the results of the examination which took 30 minutes, the charts shown in Fig. 1 illustrating the peaks a, (b), (c), d, e, (f), and (g) were obtained, the parenthesized peaks appearing only on the chart of the serum of the patient of chronic renal failure.

The mutual separation of the peaks appearing only on the chart taken on the serum of the patient of chronic renal failure and their quantification suggest the application of the quantified values of these peaks as the other values of clinical examination than the conventional values of conventional clinical examination.

For comparison, the same procedure of the highspeed liquid chromatography was carried out except that the filling of the column was carried out using the same bead-like hydroxymethylated copolymer of styrene and divinylbenzene, however, without having adsorbed or adhering the serum albumin, on the serum of the patient of chronic renal failure under dialytic treatment.

The result showed that (1)the peak a was lower than that obtained by using the filling composition of the present invention and (2) the separation of the peaksb, c, d, etc. was insufficient.

Table 1: EffluentTimeand Relative Area of Peaks of Normal Serum and Serum of Renal Failure in Liquid Chromatography

Relative Area of Peaks of Specimen - - - - abcdefg Normal Serum 9895 - - 323 52 - - Serum of 8046 130 1293 653 1001 505 38 Renal failure 11.8 Effluent time 4.7 7 6.9 7.8 8.9 10.6 25.4 of each peak to (min) 16.5 Example 2: Using the same high-speed liquid chromatograph equipped with the column filled with the filling composition obtained by the procedures shown in Example 1, the transition of the morbid state of a patient with the patient's history of from slight renal failure through hospitalization to the treatment with dialysis was followed by the chromatographic examination of the patient's serum as well as by the determination of BUN and creatinine. The results are shown in Table 2.

As is seen in Table 2, corresponding to the aggravation of the values of conventional clinical examination such as BUN and creatinine in the serum, the number of the abnormal peaks appearing in the chromatogram increased indicating the usefulness of the method according to the present invention.

Table 2: Progress of Morbid State of a Patient of Renal Failure and Results of Chromatog raphy of the Patient's Serum

Values of Relative Area of Each Peak Days of clinical examn.

observation b o d e f cc b 0 d 6 g (mg/dl) 0 72 - 938 471 - - 101 7.6 7 57 - 736 446 - - 101 8.3 48 78 116 434 514 21 116 105 9 2 aggravation 56 95 214 534 618 70 191 104 9.9 hospitalization 63 104 334 770 626 170 16 8 102 10.8 preparation for analysis 70 144 243 730 610 181 388 102 10.8 77 151 328 588 589 222 545 103 10.5 Example 3:: A female JCL-SD rat was made to be a model of the liver disorder by administration of 1,000 mg/kg of D-galactosamine and its serum was sampled before the administration (normal stage), during the morbid state and during its recovering period, and the specimens of the serum were examined by the high-speed liquid chromatograph shown in Example 1 provided with the column filled with the filling composition of the present invention. As the results, a chart shown in Fig. 2 and the relative values of area of the peaks on the chromatogram shown in Table 3 were obtained. Separately, biochemical analyses were carried out on GOT, GPT and bilirubin, the results being shown also in Table 3.

As is shown in Fig. 2 and Table 3, in the serum of the rat administered with D-galactosamine to be a model of the liver disorder, the values of GOT, GPT and bilirubin were clearly abnormal after administration as compared to the values before the administration and afterthe recovery has begun. Justcorres- ponding to the transition of the above-mentioned values, each of the peaks of more than 5 minutes of the effluent time showed a conspicuous change.

Table 3: Resultof Chromatography ofRatin Model Liver Disorder with Results of Clinical Examination

Specimen Relative Area of each Peak 0 as. Data of Clinical of serum Examination (mg/dl) 1 2 3 4 5 6 7 8 9 GOT GPT Bilirubin At ner,a1 34156 37 37 13 2311 73 - 246 128 100 70 less than state 0.3 At liver disorder 35292 41 155 30 202 38 107 512 795 4560 2260 3.9 At reco vering 37911 37 122 65 1808 78 - 59 113 540 520 0.5 state Effluent tire of each ak 4.5 6.3 6.8 7.4 8.8 peak 13.9 15.4 18.1 (min) As is possibly understood from the results of the above-mentioned examination, by the application of the high-speed liquid chromatography utilizing the filler composition of the present invention, a chromatographic pattern corresponding to the transition of the morbid state is available also in the case of the liver disorder. On the other hand, the examination of the above-mentioned specimens of the serum by the same chromatography, however, with the column filled with the bead-like oxymethylated copolymer of styrene and divinylbenzene without adsorbed or adhering the human serum albumin gave a chart in which, as is shown in the later part of Example 1,the peak 1 was lower in height and the separation of the peaks 2, 3,4,7 and 8 was insufficient.

Claims

1. A filler composition for use in a liquid chromatography column comprising beads of a hydroxymethylated copolymer of styrene and divinylbenzene, which beads have a serum protein adsorbed or adhering thereonto.

2. A filler composition according to claim 1, wherein the serum protein is albumin, globulin or a mixture thereof.

3. A filler composition according to claim 1 substantially as hereinbefore described in Example 1.

4. A process for preparing a filler composition as claimed in any one of the preceding claims, which process comprises bringing beads of a hydroxymethylated copolymer of styrene and divinylbenzene into contact with a solution of a serum protein.

5. A process according to claim 4, wherein the beads are packed in a column and the solution is passed through the column.

6. A process according to claim 4 or 5, wherein the serum protein is dissolved in water, a buffer solution, a buffer solution having a salt added thereto, or a buffer solution having an organic solvent added thereto.

7. A process according to any one of claims 4 to 6 wherein the concentration of the serum protein in the solution is from 0.2 to 5% by weight

8. A process according to claim 4 substantially as hereinbefore described in Example 1.

9. A column for use in liquid chromatography which is packed with a filler composition as claimed in any one of claims 1 to 3 or which has been prepared by a process as claimed in any one of claims 4 to 8.

10. A column according to claim 9 substantially as hereinbefore described in Example 1.

11. A method of liquid chromatography which method comprises introducing a first liquid to be analysed and a second liquid which acts as the moving phase to a column as claimed in claim 9 or 10.

12. A method according to claim 11, wherein the method is a high-speed liquid chromatographic method.

13. A method according to claim 11 or 12 wherein the first liquid is a physiological specimen.

14. A method according to claim 13, wherein the physiological specimen is blood, cerebrospinal fluid, ascites or urine.

15. A method according to claim 11 substantially as hereinbefore described in any one of the Examples.

16. A column for use in liquid chromatography which is packed with beads of a hydroxymethylated copolymer of styrene and divinylbenzene, which beads do not have a serum protein adsorbed or adhering thereonto.