GB2308079A - Purification and recycling of eluate in HPLC - Google Patents

Description

"A process for analysing a fluid" Introduction The invention relates to a process for the reverse phase chromatographic detection of analytes in a sample.

The most commonly used technique for measuring the concentration of drugs and their breakdown products in blood, plasma, serum or urine is known as high performance liquid chromatography (HPLC). This process involves the pumping of a solvent (the mobile phase), consisting of various high purity, expensive organic solvents and aqueous buffers in various combination, at high pressure (100-200 atmospheres) through a column packed with a silica based powder. As the mixture of chemicals in the plasma sample pass through this column, the various components adhere to the silica based powder to greater or lesser extents, which results in their being retained on the column for different periods of time.The result is that the various components emerge from the column separated from one another and are detected by highly sensitive devices such as ultra violet, fluorescent, or electrochemical detectors. The response of the detector (a chromatogram) is recorded and measured by an integrator and from these signals, the concentrations of the drug can be calculated from a calibration curve.

The solvent in the meantime is collected in a separate container to be disposed of as it now contains all the components which were present in the initial sample. It cannot, therefore, be recycled without contaminating the column and interfering with the next chromatogram. The initial cost of the solvent as well as the cost of disposal (equal in some countries to twice the cost of the solvent itself) is a major expense for any laboratory.

Disposal by whatever means results in environmental contamination.

However, many laboratories still recycle their mobile phase solvent. This recycling results in a build-up of contaminants and corresponding errors in all future analyses due to the appearance of negative peaks under the analyte peak. This means that when the concentration of the analyte in the recirculated mobile phase solvent is equal to that of the injected sample, no peak will be seen on a chromatogram and as a result zero will be assumed.

Similarly, when a blank (analyte-free) sample is injected onto the column, a negative peak will be detected equal in concentration to the concentration of contaminant in the recycled mobile phase.

Besides the analyte, constituents of the matrix from which the analyte has been extracted, also build-up in the mobile phase causing an increase in the background noise level, thereby rendering the system less sensitive, or can adversely affect the chromatography by saturating the active sites on the chromatography column. Low levels of analyte will be difficult to quantitate as the signal to noise ratio may be unfavourably affected. This is particularly problematical in the case of analyses using photochemical reactors (Tamoxifen) where matrix constituents are converted into fluorescent compounds which can cause severe background disturbances when recirculated.

Two possible methods of overcoming this problem have been developed by analytical chemists. The first involves the use of a large reservoir (2 to 3 litres) of mobile phase which is recirculated, the concept being that the recirculated contaminants within the mobile phase will be diluted by the large volume present and will affect the results to only a small degree. This method has two problems, namely, that the cost of the solvents used is still high and disposal of such a large volume is required. The second problem with this method is that the mobile phase is still contaminated (even though the contaminants have been diluted) and becomes more so with each sample added. Thus, although the level of contaminants is reduced, the quality of results is still affected due to the presence of the contaminants.This method has proven to be only partially successful analytically and totally unsuccessful commercially.

The second method of overcoming this problem involves the use of a mechanical switching device that sends to waste all contaminants and recirculates the uncontaminated mobile phase. This method, however, requires a costly switching valve for each machine in use and only save a portion of the mobile phase used with each sample.

It is an object of the invention to overcome at least some of the above-mentioned problems.

Statements of Invention According to the invention there is provided a process for the reverse phase chromatographic detection of analytes in a clinical sample such as blood, blood plasma, serum or urine, the process comprising the steps of : - pumping a mobile phase solvent through a column of chromatographic packing material, the mobile phase solvent being pumped from a mobile phase solvent reservoir; - pretreating the sample; - applying the pretreated sample to the column; - eluting the column with mobile phase solvent; - passing the eluate through a detection means to detect the analytes in the mobile phase solvel;t; - passing the eluate through an adsorbent column of pyrolysed particles of a polysulfonated macroporous, crosslinked, styrene-divinylbenzene polymer to remove analytes from the eluate; and - recirculating the analyte-free eluate to the mobile phase solvent reservoir.

In one embodiment of the invention the sample is pretreated in a method which includes: - activating a solid phase extraction column by washing under vacuum first with methanol and then with 0.1 M hydrochloric acid; - applying the sample, 0.1 M hydrochloric acid and an internal standard solution (.00048% Flufenamic acid) to the column; - eluting the column under vacuum to dryness to remove extraneous matter; - washing the column with water to remove any remaining extraneous matter; and - eluting the pretreated sample from the column with a solution of methanol/acetonitrile in a ratio of 60:40 (v/v).

In a further embodiment of the invention, the sample is pretreated in a method which includes: - mixing the sample with 50% urea in a ratio of 1:1 (v/v); - vortexing the mixture for 10 seconds before letting the mixture rest for 5 minutes; - adding excess diethylether; - rotating the mixture in a test tube rotator for 10 minutes at 80 r.p.m.; - centrifuging the mixture at lOOOg for 3 minutes to separate aqueous and ether phases; - placing the aqueous and ether phases in an alcohol bath at -35 C to freeze the aqueous phase; - transferring the ether phase to a tube and evaporating to dryness at 400C in a stream of air to leave the pre treated sample; and - reconstituting the pre-treated sample.

In a still further embodiment of the invention, the sample is pretreated in a method which includes: - mixing the sample with a 1:1 mixture of methanol/acetonitrile in a ratio of 2:5 (v/v); - vortexing the mixture for 10 seconds; - centrifuging the vortexed mixture at 3000g for 3 minutes to produce a supernatant; and - removing the supernatant which comprises the pretreated sample.

Preferably the analytes are pharmaceuticals or pharmaceutical breakdown products.

Ideally, the reverse phase chromatography is reverse phase high performance liquid chromatography.

Most preferably the chromatographic packing material is octadecylsilane.

Typically the flow rate through the adsorbent column is approximately 1 ml/min.

Detailed Description of the Invention The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the accompanying drawings, in which: Fig. 1 is a block diagram illustrating the process according to the invention; Fig. 2 is a perspective view of an adsorbent column used in the process of the invention; Fig. 3 is a perspective view with a portion cut-away of the adsorbent column of Fig. 2; Fig. 4 is a chromatogram obtained from Example 1; Fig. 5 is a chromatogram obtained from Example 2; Fig. 6 is a chromatogram obtained from Example 3; Fig. 7 is a chromatogram obtained from Example 4; Fig. 8 is a chromatogram obtained from Example 5; Fig. 9 is a chromatogram obtained from Example 6; Fig. 10 is a chromatogram obtained from Example 7;; Fig. 11 is a chromatogram obtained from Example 8; and Fig. 12 is a chromatogram obtained from Example 9.

Referring to the drawings and initially to Fig. 1 thereof, there is illustrated a process according to the invention for the reverse phase chromatographic detection of analytes in a sample, the process being indicated generally by the reference numeral 1. Generally, the process involves the use of high performance liquid chromatography (HPLC) and includes the initial step of packing a HPLC column 2 with a HPLC packing material such as octadecylsilane (ODS). A mobile phase solvent 4 is then pumped by means of a high pressure HPLC pump 5 from a mobile phase solvent reservoir 6 along a feed line 7 and through the HPLC column 2.

The sample is applied to the column by means of a sample injector 10 which injects the sample into the mobile phase solvent passing through the feed line 7 between the HPLC pump 5 and the HPLC column 2. As the HPLC column 2 is eluted with mobile phase solvent, the eluate is fed along an eluate feed line 11 to a detecting means 12 which detects the analytes in the eluate. Depending on which analyte is being detected, the detecting means may be an ultraviolet, fluorescence or electrochemical detector.

The response of the detector is recorded graphically in the form of a chromatogram and measured by an integrator (not shown).

The eluate is then pumped along a post detector feed line 15 to an adsorbent column 16 which contains a carbonaceous adsorbent 17 comprising pyrolysed particles of a polysulfonated, macroporous, crosslinked, styrenedivinylbenzene polymer, sold under the trade name AMBERSORBN. As the eluate is pumped through the adsorbent material 17, at least 99% of the analytes in the eluent are retained by the material 17, thus leaving a substantially pure mobile phase solvent which is recirculated to the mobile phase solvent reservoir 6.

Thus, the recirculated mobile phase solvent can be reused without adversely affecting a following chromatographic analysis.

In more detail and referring to Figs. 2 and 3, the adsorbent column 16 comprises an elongated, hollow cylinder 20 with a base 21 and a top 22. A connecting nozzle 23 on the top 22 facilitates connection of the post detector feed line 15, with a similar nozzle 24 on the base 21 feeding into the mobile phase solvent reservoir 6.

The bed of adsorbent material 17 is supported within the column 16 between a plug of polypropylene material 28 which itself rests on the base 21, and a pad 29 of similar material.

The process of the present invention is particularly applicable for the detection of analytes in biological samples such as blood, blood plasma, serum and urine. In this respect, analytes particularly suitable for detection by the process of the invention are pharmaceuticals and pharmaceutical metabolites contained in the biological samples. These analytes include, for example, Amoxycillin, Ampicillin, Antipyrine, Bromazepam, Chloroquine, Diclofenac, Flucloxicillin, Flurbiprofen, Ketoprofen, Mefenamic acid, Metformin, Metoclorpromide, Methylprednisolone, Naproxen, Paracetamol, Piroxicam, Sulphamethizole, Sulpiride, Tamoxifen, Theophylline, and Timolol.Generally, in order to facilitate the detection of the desired analyte, the sample is first treated to remove extraneous matter, which in the case of blood, would include, for example, blood cells, enzymes and other structural and functional proteins. Depending on which analyte is to be detected, the sample is usually pretreated using one of three techniques, namely, protein precipitation, solvent extraction or solid phase extraction. Each of these techniques are described in more detail below.

Generally, a clinical study involves up to 1000 separate assays. Heretofore, in order to keep the accuracy of the results as high as possible, fresh mobile phase solvent was required for each batch of samples (+ 50 samples/ batch). Obviously, the expense involved in supplying such amounts of solvent is enormous. However, with the implementation of the process of the present invention, and in particular the use of the carbonaceous adsorbent material as described herein to remove contaminating analytes from the mobile phase, the mobile phase can be recirculated without affecting the quality, accuracy or repeatability of the results. A further advantage with this improved process is that, due to the fact that the mobile phase does not have to be changed during a clinical study, the chances of it being contaminated by airborne contaminants are kept to a minimum.The time required to prepare the mobile phase is also eliminated.

The following examples illustrate the process of the invention.

Detection of Diclofenac Extraction Procedure (Solid Phase Extraction) A C18 bond elut solid phase extraction column was activated by first washing to dryness with 1 ml methanol following by washing with 1 ml 0.1 M hydrochloric acid, drawing each through by vacuum. 0.5 mls of 0.1 M hydrochloric acid, 200 z1 of plasma and 200 z1 internal standard solution (containing 97.5 ng flufenamic acid) were then added to the column reservoir. These components were drawn through the column under vacuum until the column was dry whereupon the column was washed, again to dryness, with 1 ml of water, thus leaving the analyte, in this case Diclofenac, and the internal standard bound to the column with most of the extraneous matter washed off.

The diclofenac was eluted with 300 > 1 of a methanol/acetonitrile (3:2) solution. The eluate was then diluted with 300 1 of water before being vortexed for 10 seconds.

Example 1 100 g1 of a solution of Diclofenac in mobile phase (63 zg/ml) was injected onto the HPLC column (Brownlee, Spheri-5, RP-18, 5U, 100 x 4.6 mm). Reversed phase chromatography was performed using a mobile phase consisting of methanol/acetonitrile/ phosphate buffer (0.067M, pH 6.00) in a ratio of 28.8:19.2:52.0 (V/V) at a flow rate of 1 ml per minute at ambient temperature. The final pH of the mobile phase was adjusted to 7.00 with 1 M sodium hydroxide. The detection means comprised a W spectrophotometer operating at 275 nm.

A chromatogram of the results is illustrated in Fig. 4.

Example 2 The protocol of Example 1 was repeated except that the mobile phase contained Diclofenac in a concentration similar to the Diclofenac solution used in Example 1.

A chromatogram of the results is illustrated in Fig. 5.

Example 3 The protocol of Example 1 was repeated with the exception that the sample injected onto the column contained zero Diclofenac.

A chromatogram of the results is illustrated in Fig. 6.

Example 4 The protocol of Example 1 was repeated with the exception that the injected sample was that prepared in the extraction procedure described above.

A chromatogram of the results is illustrated in Fig. 7.

Referring now to the chromatogram illustrated in Fig. 4 (Example 1), a peak characteristic of Diclofenac can be clearly seen indicated by the arrow A.

Referring now to the chromatogram illustrated in Fig. 5 (Example 2), the problems of analyte contamination in the mobile phase solvent is illustrated. Thus, even though Diclofenac is present in the injected sample, no peak is seen due to the presence of an equal concentration of analyte in the mobile phase solvent. Similarly, the chromatogram illustrated in Fig. 6 (Example 3) demonstrates the problems associated with injecting a blank (analyte-free) sample onto the column when the mobile phase contains analyte. Thus, a negative peak equivalent to the concentration of Diclofenac in the mobile phase is detected.

The chromatogram illustrated in Fig. 7 (Example 4) shows the detection of Diclofenac in an extracted plasma sample.

The peak marked with the arrow B indicates Diclofenac while the peak marked with the arrow C indicates the internal standard solution.

Detection of Tamoxifen Extraction Procedure (Solvent Extraction) 1 ml of 50% urea was added to 1 ml of plasma in a 10 ml cap culture tube. After vortexing for 10 seconds, the mixture was left for 5 minutes whereupon 5 mls of diethylether was added. After rotating for 10 minutes in a test tube rotator (80 r.p.m.), the extract was centrifuged at 1000 g for 3 minutes. The tubes were left in an alcohol bath set at -350C for three minutes, causing the aqueous layer to freeze. The ether layer was transferred to a 5 ml glass ampoule and evaporated to dryness at 400C in a stream of air. The residue was redissolved in mobile phase.

Example 5 (Blank Sample) 50 81 of a blank sample was injected onto a HPLC column (Metachem Inertsil ODS-2, 5U, 150 x 4.6 mm). Reversed phase chromatography was performed using a mobile phase consisting of acetonitrile and phosphate buffer (0.067 M, pH 2.20) in a ratio of 50:50 at a flow rate of 1 ml per minute at ambient temperature. The final pH of the mobile phase was 2.94. A post-column fluorescence activator was installed consisting of 1 M x 0.3 mm knitted teflon tubing held on a metal frame 2 cm above an 8 watt 30 cm W 254 nm germicidal lamp (silvania G8T5). Analytes were detected by a fluorescence detector (Perkin, Elmer LS 40) set at 260 mm (excitation 10 nm slit) and 365 nm (Emission, 10 nm slit).

A chromatogram of the results is illustrated in Fig. 8.

Example 6 The Tamoxifen sample prepared in the Tamoxifen extraction procedure described above was reconstituted in 200 Cil mobile phase. 50 1 of this solution was injected onto a HPLC column and the protocol of Example 5 was carried out thereafter.

A chromatogram of the results is illustrated in Fig. 9.

Example 6 was then repeated nine times with the mobile phase being recirculated each time without being passed through an adsorbent column.

Example 7 The protocol of Example 5 was repeated with the exception that the mobile phase solvent used was that which was used nine times in Example 6. However, after passing through the detector, the eluate was passed through an adsorbent column and stored for use in Example 8.

A chromatogram of the results is illustrated in Fig. 10.

Example 8 The protocol of Example 5 was carried out with the exception that the mobile phase used was recirculated from Example 7.

A chromatogram of the results is illustrated in Fig. 11.

Referring now to the chromatogram illustrated in Fig. 8 (Example 5), a normal base line indicative of a blank sample is clearly illustrated, the mobile phase used in this example was fresh mobile phase and thus contained no contaminating analyte.

Referring now to Fig. 9 (Example 6), the characteristic chromatogram of Tamoxifen and its metabolites is clearly illustrated. The peak marked by the arrow B indicates the presence of Tamoxifen. The other peaks relate to Tamoxifen metabolites. Again, the mobile phase used in this example was fresh mobile phase and contained no contaminating analytes.

Referring now to the chromatogram illustrated in Fig. 10 (Example 7), a blank sample containing no Tamoxifen was analysed using a mobile phase solvent which had been used in ten analytical procedures similar to Example 6. It can be clearly seen from this chromatogram that the build up of Tamoxifen analytes in the mobile phase and matrix constituents which have been converted into fluorescent compounds has resulted in negative peaks. However, the chromatogram illustrated in Fig. 11 (Example 8) clearly shows the normal base line indicative of a blank sample.

This is due to the recirculated mobile phase from Example 7 being recirculated through an adsorbent column before being used as the mobile phase solvent in Example 8.

Detection of Fenofibric Acid Extraction Procedure (Protein Precipitation) To 200 ml of plasma, 500 z1 of a 1:1 mixture of methanol/acetonitrile was added. After vortexing for 10 seconds, the mixture was centrifuged at 3000 g for 3 minutes.

Example 9 A 50 p1 aliquot of the resultant supernatant prepared in the Fenofibric acid extraction procedure described above was directly injected onto the analytical column (Waters Novapak C18, 4U, (150 x 3.9 mm). Reverse phase chromatography was performed using a mobile phase consisting of methanol, acetonitrile and phosphate buffer (0.067 M, pH 6.00) in the ratio of 20:22:58, at a flow rate of lml.min at ambient temperature. The detection means comprised a UV spectrophotometer operating at 275 nm. The eluate was recirculated to a mobile phase solvent reservoir through an adsorbent column.

A chromatogram of the results is illustrated in Fig. 12.

The peak marked by the Arrow C represents Fenofibric acid.

The peak marked by the Arrow D represents any extraneous matter left in the plasma after the extraction procedure.

The invention is not limited to the embodiments or examples as given above but may be varied in detail.

Operational parameters and sequences may also be varied without departing from the spirit of the invention.

Claims (12)

1. A process for the reversed phase chromatographic detection of analytes in a clinical sample such as blood, blood plasma, serum or urine, the process comprising the steps of: - pumping a mobile phase solvent through a column of chromatographic packing material, the mobile phase solvent being pumped from a mobile phase solvent reservoir; - pretreating the sample; - applying the pretreated sample to the column; - eluting the column with mobile phase solvent; - passing the eluate through a detection means to detect the analytes in the mobile phase solvent; - passing the eluate through an adsorbent column of pyrolysed particles of a polysulfonated macroporous, crosslinked, styrene-divinylbenzene polymer to remove analytes from the eluate; and - recirculating the analyte-free eluate to the mobile phase solvent reservoir.

2. A process as claimed in claim 1 in which the sample is pretreated in a method which includes: - activating a solid phase extraction column by washing under vacuum first with methanol and then with 0.1 M hydrochloric acid; - applying the sample, 0.1 M hydrochloric acid and an internal standard solution (.00048% Flufenamic acid) to the column; - eluting the column under vacuum to dryness to remove extraneous matter; - washing the column with water to remove any remaining extraneous matter; and - eluting the pretreated sample from the column with a solution of methanol/acetonitrile in a ratio of 60:40 (v/v).

3. A process as claimed in claim 1 in which the sample is pretreated in a method which includes: - mixing the sample with 50% urea in a ratio of 1:1 (v/v); - vortexing the mixture for 10 seconds before letting the mixture rest for 5 minutes; - adding excess diethylether; - rotating the mixture in a test tube rotator for 10 minutes at 80 r.p.m.; - centrifuging the mixture at 1000g for 3 minutes to separate aqueous and ether phases; - placing the aqueous and ether phases in an alcohol bath at -350C to freeze the aqueous phase; - transferring the ether phase to a tube and evaporating to dryness at 400C in a stream of air to leave the pre-treated sample; and - reconstituting the pre-treated sample.

4. A process as claimed in claim 1 in which the sample is pretreated in a method which includes: - mixing the sample with a 1:1 mixture of methanol/acetonitrile in a ratio of 2:5 (v/v); - vortexing the mixture for 10 seconds; - centrifuging the vortexed mixture at 3000g for 3 minutes to produce a supernatant; and - removing the supernatant which comprises the pretreated sample.

5. A process as claimed in any of claims 1 to 4 in which the analytes are pharmaceuticals or pharmaceutical breakdown products.

6. A process as claimed in any of claims 1 to 5 in which the reverse phase chromatography is reverse phase high performance liquid chromatography.

7. A process as claimed in any preceding claim in which the chromatographic packing material is octadecylsilane.

8. A process as claimed in any of claims 1 to 7 in which the flow rate through the adsorbent column is approximately 1 ml/min.

9. A process substantially as hereinbefore described with reference to Examples 1 to 4.

10. A process substantially as hereinbefore described with reference to Examples 5 to 8.

11. A process substantially as hereinbefore described with reference to Example 9.

12. A process substantially as hereinbefore described with reference to the accompanying drawings.