GB2348380A - Sampling apparatus with silicone rubber membrane - Google Patents

Abstract

Apparatus for sampling an organic substance, eg tolbutamide, from a test liquid 7, eg urine, comprises a main reservoir 1 into which is dipped a second reservoir in the form of a tube 2 sealed at its lower end by a silicone rubber membrane. The second reservoir, containing a suitable liquid extractant, is dipped into the test liquid for sufficient time for the required substance to diffuse into the extractant, which is then removed and tested conventionally.

Description

SAMPLING APPARATS AND METHOD OF SAMPLING USING SAME This invention relates to an apparatus and a method for sampling organic molecules from a test solution containing them. More particularly, the invention is directed to the extraction of organic molecules, in particular, though not exclusively, for use in the analysis for one or more organic molecular species of biological fluids such as urine, blood, plasma and saliva. This is an important technique since it may be used for example for monitoring levels of biologically active and pharmaceutical species.~ Silicone rubber membranes are known for use in separating organic molecules from fluids, but at present the known apparatuses utilising these membranes are somewhat crude, cumbersome and expensive. One example of such an apparatus is disclosed in published UK patent application GB-A-2207064. In this apparatus the silicone rubber membrane, which is non-porous, is sandwiched between two plates, with each plate being recessed to define respective chambers on opposite sides of the membrane for accommodating test and receptor solutions. The test and receptor solutions are pumped in from opposite sides of the plates such that they flow in the same direction on either side of the membrane so that the organic molecules of interest are able to diffuse through the membrane from the test solution into the receptor solution which can then be analysed for the organic species in question.

A further method which is widely used in the extraction of pharmacologically active molecules from biological fluids is the use of a pellicular packing in a glass, metal or plastic column, known as a solid phase extraction column. The test solution is passed through the column and the organic molecules of interest adhere to or are adsorbed on the packing. The column is then washed several times with the solvent of the test solution to ensure that all of the sample that has not been immobilised on the packing is washed clear of the column. A different solvent is then used to wash the organic molecules of interest from the packing and these washes are then collected and passed for analysis. This is a timeconsuming process, however, which involves using considerable volumes of solvent and although it may be automated, it is expensive. This technique also has limited specificity in terms of the efficiency with which the organic species are extracted from the test solution.

It is also known to extract amines from waste waters using porous membranes in which the pores have been filled with an organic solvent or oil, as is described for example in G. Audunsson, Anal. Chem., 1986,58,2714-2723. Because the organic solvent or oil is only physically held by the pores, there is often a problem as regards adequate retention of the solvent or oil, requiring maintenance of equal pressures on both sides of the membrane at all times.

This is difficult to achieve and implement in a practically useful arrangement.

An object of the present invention is to provide a new, cheaper and easier to use apparatus and associated method for effecting the transfer of one or more organic molecular species from a test solution to a receptor solution for subsequent analysis. It is a further object of the invention to provide such an apparatus and method which lend themselves readily to automation.

With this object in view, the present invention provides, in a first aspect, an apparatus for sampling one or more species of organic molecules from a test solution, the apparatus comprising: (i) a first solution reservoir including a window therein sealed by a non-porous silicone rubber membrane; and (ii) a second solution reservoir containing at least that portion of the first solution reservoir which includes the sealed window.

As used herein, the term"non-porous"as applied to the silicone rubber membrane means that the membrane is substantially devoid of holes or passageways allowing normal physical migration of the relevant organic molecules from one side of the membrane to the other. However, the membrane has a molecular structure such that the organic molecular species of interest dissolve in the material of the membrane and move thereacross by diffusion within the structure of the membrane material on a molecular scale.

Thus, for the extraction of the molecular species from the test solution, the membrane does not rely on molecular size, as is the case with conventional filter or dialysis membranes.

In preferred embodiments of the apparatus of the invention the non-porous silicone rubber membrane is of dimethylpolysiloxane. Examples of such silicones suitable for use in forming the membranes for use in the invention are commercially readily available, for example as Dow Corning's products (R) 732 Multi-purpose Sealant (clear), (R) 3145 MIL-A-46416 Adhesive/Sealant (clear), and (R) 3140 Coating.

For use in this invention, the silicone rubber membrane may be premanufactured, for example if such membranes having suitable thicknesses and other characteristics are commercially available, but in the context of the currently most preferred manner of carrying out the invention, the silicone membrane is prepared specially, from, for example, any of the above exemplary silicone products.

By way of example, a membrane suitable for use in the invention may be prepared by dissolving an appropriate amount of the silicone product in an organic solvent such as toluene, preferably with the use of stirring to aid dissolution and with sonication to remove air bubbles. The solution is left to stand for a period of the order of a few to several minutes and then it is poured onto a glass plate which has been pretreated with a surface coating of polytetrafluoroethylene (PTFE). The PTFE-coated glass plate may be made by sticking onto a glass sheet adhesivebacked PTFE available commercially for example from Tygovac/Tygofluor. Using a motorised blade or equivalent mechanical device, the silicone rubber solution is spread thinly across the plate to form a uniform thin film, for example with a thickness in the range of from about 20 to about 50 ssm, more preferably a thickness in the region of around 25 Sm. The cast film is allowed to dry, e. g. overnight in a fume cupboard, to allow the toluene or other solvent to evaporate, and during this period moisture in the atmosphere typically causes the silicone to cure and crosslink, resulting in a coherent silicone rubber film which can then be peeled away from the PTFE-coated plate, cut to the required size and attached over the window of the first solution reservoir of the apparatus.

The above is just one exemplary manner in which the silicone rubber membrane for use in the invention may be made, but of course, other techniques for membrane preparation, for example extrusion, may alternatively be used.

In preferred embodiments of the apparatus of the invention the first solution reservoir is that which contains the sample, particularly biological sample, containing the species to be extracted, and the second solution reservoir is that containing the receptor solution into which the species are extracted and preferably subsequently sampled therefrom for analysis purposes.

However, it is still within the scope of the invention for the solutions to be the other way around, namely the test solution for sampling to be within the second (i. e. larger) reservoir and the receptor solution to be within the first (i. e. smaller) reservoir. However, the former arrangement is preferred since it lends itself to greater flexibility in the subsequent handling of the extracted sample.

In preferred practical embodiments of the apparatus of the invention the first solution reservoir is in the form of a hollow tube, e. g. of glass or of plastics, having an open lower end forming the window which is sealed by the silicone membrane. The opposite, also open, end of the thus sealed tube provides a mouth by which the tube can be filled or emptied with solution. The membrane is preferably secured in a sealing fashion to the open lower end of the tube by for example adhesive tape or any other suitable securing means.

In preferred practical embodiments of the invention the second solution reservoir takes the form of a vial or similar receptacle capable of containing at least that portion of the first solution reservoir which includes its sealed window. That degree of containment by the second solution reservoir should be such that the window sealed with the silicone membrane extends far enough into and towards the base of the second solution reservoir such that it will be immersed in an amount of solution placed in the second solution reservoir for the purpose of sampling.

In practical embodiments of the apparatus of the invention there are also provided support means associated with either or both of the first and second reservoirs for enabling the first solution reservoir (i. e. hollow tube in preferred embodiments) to be suspended within the second solution reservoir (i. e. vial in preferred embodiments), so that the sealed window is maintained in a substantially fixed location with respect to the second solution reservoir, whilst the sampling/extraction takes place.

Given the essential nature to the invention of the first solution reservoir with its sealed window, it is to be understood that the present invention independently provides, as a second aspect, the first solution reservoir alone as defined above in the context of the definition of the first aspect of the invention, i. e. not necessarily in combination with the defined second solution reservoir.

Thus, in accordance with this second aspect of the invention, there is provided an apparatus for use in sampling one or more species of organic molecules from a test solution, the apparatus comprising a solution reservoir including a window therein sealed by a non-porous silicone rubber membrane. Preferred forms of this solution reservoir are as discussed above in the context of the first solution reservoir of the apparatus of the first aspect of the invention.

The present invention further provides, in a third aspect, a method for sampling one or more species of organic molecules from a test solution containing same, using the apparatus according to the first aspect of the invention. Thus, the method according to this third aspect comprises: (i) providing an apparatus according to the first aspect of the invention; (ii) placing the test solution in the first solution reservoir; (iii) placing the receptor solution in the second solution reservoir; (iv) inserting the first solution reservoir into the second solution reservoir such that the latter contains at least that portion of the first solution reservoir which includes the sealed window and such that the sealed window is immersed in the receptor solution in the second solution reservoir; (v) optionally, allowing the apparatus to stand for a predetermined period of time; and (vi) removing at least a sample of the receptor solution from the second solution reservoir for subsequent analysis for the desired organic molecular species.

In the above method aspect of the invention, it is especially preferred that the test solution comprising the biological fluid for sampling is that which is placed in the first solution reservoir (i. e. hollow tube in preferred embodiments), since that arrangement lends itself particularly well to subsequent handling of the extracted sample in the second solution in its respective reservoir, and it also lends itself well to a system in which multiple sampling apparatuses are provided for carrying out large numbers of simultaneous sampling under the control of an automation system. However, it is still possible within the scope of the invention for the test solution to be placed in the second solution reservoir (i. e. vial, in preferred embodiments) and the receptor solution in the first solution reservoir.

Incidentally, the terms"test solution"and"receptor solution"as used herein are intended to mean solutions based on any solvent or combination of solvents, with or without impurities or other solvents therein, apart from the organic molecular species which are desired to be sampled. Commonly the receptor solution will be aqueous, but alternatively it could be an alcohol or any other suitable solvent system. The test solution may be the biological fluid per se, or alternatively it may be an aqueous, organic or even aqueous/organic solution of a sample of that biological fluid.

It is presently intended that the volume of the second solution reservoir and the size of the sealed window of the first solution reservoir will be of a reasonably small size such that a volume of test solution of for example up to about lml is sufficient for placing in the first solution reservoir to cover the sealed window when it is placed in the second solution reservoir. However, any suitable greater volume of test solution may be used, if desired or if necessary. Similarly, the amount of receptor solution which is placed in the second solution reservoir may be selected to provide a desired volume of solution for subsequent analysis work. Typically, however, about 100p1 of receptor solution will be suitable.

In the method of the invention, prior to the first solution reservoir (containing its respective solution) being placed into the second solution reservoir (containing its respective solution for beginning the extraction process), if appropriate or if necessary the pH of the test solution may be adjusted by addition of an appropriate amount of acid or base, as will be well known to persons skilled in the art. This will apply in cases where it is necessary to establish a particular pH gradient between the test solution and the receptor solution in order to drive the particular organic molecular species of interest into and across the membrane. Establishing such a pH gradient will generally be necessary or at least preferable when the organic species to be extracted from the test solution is ionizable. However, with organic molecules of interest which are not ionizable a differential in ionic strength between the test and receptor solutions will be appropriate, which can be established for example by addition to the test solution of an appropriate amount of a salt, such as sodium chloride. Such techniques are within the common general knowledge of the persons skilled in the art.

In the present invention it will generally be the case that the organic species of interest are only able to pass through the non-porous silicone rubber membrane as a neutral species. The organic molecules dissolve in the matrix of the membrane and then proceed through it by diffusion. Thus, when the method of the invention is applied for example to a carboxylic acid which is tombe extracted, an excess of mineral acid (e. g. hydrochloric acid) is preferably added to the test solution to prevent dissociation of the carboxylic acid species.

Correspondingly, when the method of the invention is applied for example to an amine as the molecular species of interest to be extracted, a base (e. g. sodium hydroxide) is preferably added to prevent dissociation of the amine.

By way of example, therefore, coupled with the above discussion of pH gradient to force the organic molecules of interest into and across the membrane, a typical set-up for the extraction of a carboxylic acid species will involve the test solution containing an excess of acid, with the receptor solution being alkaline to establish the necessary pH gradient, whereas in the case of an amine as the molecular species to be extracted, the test solution will contain excess base, with the receptor solution being acidic to establish the necessary pH gradient.

If desired, in the carrying out of the method of the invention the receptor solution may typically comprise water, an organic solvent and also a buffer. The aqueous organic solvent mixture will be selected such that it is a good solvent for the organic species of interest o be extracted. Examples and concentrations of organic and aqueous solvent components will be selected according to the skilled person's general technical knowledge in the art.- In practical terms, in the carrying out of the method of the invention, once the two solution reservoirs have been brought together in the manner defined with the sealed window of the first solution reservoir immersed in the solution contained within the second solution reservoir, the arrangement is preferably left to stand for a predetermined period of time to allow the organic molecules of interest to diffuse through the membrane and into the receptor solution, this period being sufficient to establish a sufficient concentration of the organic species in the receptor solution for subsequent detection and/or analysis. Typically, this rest period will be for a matter of, say, 10 minutes to 1 hour, perhaps typically around 30 minutes, and typically at a temperature of room temperature or somewhat above, e. g. around 45 C (for example by virtue of the apparatus being immersed in a water bath held at the required temperature).

Typically the method of the present invention may be carried out using an internal standard substance which is added to the sample before the extraction procedure begins.

Suitable internal standard substances will usually be chemical compounds which are structurally similar to the organic molecular species to be sampled. The concentration of internal standard added to the sample will normally be selected so as to give an acceptable response signal during the post-extraction analysis. During the extraction the internal standard behaves in substantially the same manner as the organic species of interest in terms of its dissolving in and migration through the non-porous membrane, thereby enabling the subsequent analysis to be corrected for any deficiencies in the extraction mechanism.

Suitable internal standard materials for any given organic species of interest will be readily apparent or determinable by persons skilled in the art.

Once the extraction procedure is complete, the receptor solution, or a sample of it, is removed from the reservoir containing it such as by use of a pipette or any other suitable piece of laboratory equipment. The solution can then be taken for analysis for detection and/or quantification of the organic molecular species (which may be one or more different organic species) of interest and the internal standard (if used). Typically such analysis may be by mass spectrometry or liquid chromatography-mass spectrometry although any other analytical method, e. g. other forms of chromatography, W spectroscopy, NMR and such like may also be used.

Preferred embodiments of the present invention in its various aspects will now be described in further detail, with reference to the accompanying drawings, in which: Fig. 1 is a schematic side, part-sectional view of an exemplary sampling apparatus according to the first aspect of the invention; Fig. 2 is a schematic representation of an example of the manner of implementation of the extraction method of the invention, as described in Example 1 below; Fig. 3 is a graph showing the results of a series of runs of an extraction experiment described in detail in the Example further below; and Fig. 4 shows a typical chromatogram obtained from the analysis procedure exemplified in Example 1 below.

Referring to Figure 1, the sampling apparatus, represented here in its simplest practical form comprising a single test solution reservoir and a single receptor solution reservoir, comprises a vial 1 as the receptor solution reservoir, which is preferably a cheap disposable glass vial, and an open-ended glass or clear plastic tube 2. The lower end of the tube 2 is sealed with a membrane 3 of non-porous silicone rubber.

The membrane 3 was prepared by dissolving 4 grams of Dow Corning (R) 3140 silicone in 25ml of toluene at room temperature, with stirring and sonication for 15 minutes to fully dissolve the silicone and to remove air bubbles. The solution was then allowed to stand for ten minutes, to form a clear, colourless solution. The solution was then spread onto a PTFE-coated glass plate using a blade to form a uniform thin film with a thickness of approximately 25um.

It was then allowed to dry overnight in a fume cupboard, during which time the toluene solvent evaporated and the film cured and crosslinked to form a coherent silicone rubber membrane which could then be peeled away from the glass plate and cut to an appropriate size to prepare a patch of membrane for securing to the open lower end of the glass tube 2 of the above described apparatus.

The membrane 3 was secured to the tube using a wrapping of adhesive tape 4.

To assemble the apparatus ready for the extraction process the tube 2 with its open end sealed by the membrane 3 is inserted into the vial 1 via its mouth 6. Into the tube 2 has previously been placed approximately lml of test solution, the tube 2 being inserted far enough into the vial 1 so that the open end and the attached membrane 3 are beneath the surface of the receptor solution 7. Any suitable form of support means for anchoring the tube 2 in this configuration with respect to the vial 1 can be employed, which in this simplest practical form can be a bung 5 or other similar packing means.

An amount (e. g. typically 100ul) of receptor solution is placed into the vial 1 and then the apparatus is left for a suitable period of time for the extraction of the desired organic molecular species to take place. Once that is complete, the tube 2 can be removed from the vial 1 and the receptor solution removed or sampled for subsequent analysis.

It is envisaged within the scope of the present invention that the sampling apparatus can be worked up into a multi-sampling array, in which a relatively large number of sampling tubes 2 can be employed with a single, larger test solution reservoir, so that multiple extraction runs can be carried out simultaneously. Such large-scale automation of sampling procedures are of course well known in the art, so the principles and constructional details of which will not be described further here.

To further illustrate the present invention and its effectiveness for accurate sampling of organic species, a series of experiments was carried out on the extraction of the drug tolbutamide from urine, according to the following Examples.

Example 1.-Calibration curve generation.

Twenty 1 ml volumes of urine collected from healthy human male volunteers were aliquotted into vials. To each vial was then added different amounts of tolbutamide (ex Sigma Ltd, Poole, Dorset, UK, Lot no. 16H0898, 100% purity). For the amounts of tolbutamide added see Table 1, and the notes to it, below. Each aliquot sample was then spiked with chlorpropamide as internal standard (ex Sigma Ltd, Poole, Dorset, UK, Lot no. 31H0722, 99.9% purity). The chlorpropamide was prepared as a methanolic solution containing 14.76 Hg/ml and 25S1 of the solution was added to each sample.

Each sample was then subjected to an extraction procedure in accordance with the procedure shown in Fig. 2 of the accompanying drawings, using the extraction apparatus as described above with reference to Fig. 1 of the accompanying drawings.

A typical chromatogram as obtained from this analysis procedure is illustrated, by way of example only, in Figure 4 of the accompanying drawings.

For each run of the experiment described above the theoretical concentration of tolbutamide in the extracted receptor solution was calculated on the basis of the measured concentrations of tolbutamide and internal reference standard in the initial urine sample.

The chromatographic system, settings and conditions for the analysis were as follows: Svstem Hewlett Packard HPLC Conditions Injection volume: 5U1 Draw position: 0.5mm Draw speed: 200min Eject speed: 200min Flow rate: 0.20 ml/min-1 Mobile phase: Mixture of 700ml 0.002M ammonium formate buffer and 300ml acetonitrile (adjusted to pH3 with formic acid) Stop time: 13.0 min Analytical column: 100 x 2.1 mm, BDS Hypersil 3m.

[I. D. No. 456] Column Temperature: 40 C Divert Valve Timetable: Initial : column switch on, contact 1 off 0.00 min : column switch on, contact 1 on 0.10 min : column switch on, contact 1 off 0.13 min : column switch off, contact 1 off 2.00 min : column switch on, contact 1 off Retention times (approximate) Internal Standard 8.0 min Tolbutamide 11.2 min LC/MS/MS Conditions Source Capillary voltage: 3.20 Extractor: 3 RF lens: 0.11 Source block temperature: 100 C Desolvation temperature: 450 C Analyser LM resolution 1: 15.0 HM resolution 1: 15.0 I energy 1: 0.5 Entrance: 0 Exit: 0 LM resolution 2: 15. 0 HM resolution 2: 15.0 I energy 2: 1.8 Multiplier: 650 Gas Flows Nebuliser: 70 litres/hour Desolvation: 735 litres/hour Analytes Internal Standard (M) +=277.25, daughter = 59.70.

Scan Time=5.5-10.0 min.

Collision Energy=25 eV Cone = 24V Tolbutamide (M) +=271.30, daughter = 73.80 Scan Time=10.0-13.0 min. Collision Energy=20 eV Cone = 24 V After each run the characteristic peak area indicative of the tolbutamide present in the extracted receptor solution was measured and from this was calculated the actual concentration sampled in the receptor solution. The results are shown in Table 1 below.

Table 1

Calibrator Theometrical Peak IS Ratio Calculated Residual C. V.

Concentration Area Area Observed Cone lSl 4. 96 414 14620 2. 832E-02 4. 34-12. 92% 2.09% 1S2 4. 96 265 9086 2. 917E-02 4. 51-8.97% 2S1 13.70 614 7241 8.479E-02 15.71 14.64% 16.15% 2S2 13. 70 314 4658 6. 741E-02 12. 21-10.89% 3S1 31. 89 2178 12609 1. 727E-01 33. 39 4. 72% 0.13% 3S2 31. 89 2029 1176S 1. 724E-01 33. 33 4. 52% gS1 57. 71 2147 7919 2. 711E-01 53. 19-7.84% 13.28% 4S2 57. 71 3051 9321 3. 273E-01 64. 49 11. 75% 5S1 9Z. 64 5031 10066 4. 998E-01 99. 19 7. 07% 5.99% 5S2 92.64 2488 5418 4.592E-01 91. 02-1.75% 6S1 135. 00 4118 5765 7. 143E-01 142. 34 5. 43% 0.56% 6S2 135. 00 10361 14390 7. 200E-01 143. 48 6. 28%

7S1 189.00 10101 8907 1.134E+00 226.77 19.98% 28.80% 7S2 189.00 14041 18714 7.503E-01 149. 58-20.86% 8S1 249. 75 9837 7575 1. 299EiOO 259. 87 4. 05t 4. 31@ 8S2 249.75 17219 14093 1.222E+00 244. 42-2.13% 9S1 324. 00 18512 11561 1 601E+00 320. 75-1.00% 7.04% 951 324. 00 16874 9538 1. 765E+OO 354. 52 9. 42k 10S1 398.25 19472 10309 1.889E+00 378. 60-4.93% 1.40% 10S2 398.25 24896 12922 1.927E+22 386. 21-3.02% In the above Table 1 the column headings have the following meanings: Calibrator: there are 20 calibrators, the different concentrations of tolbutamide with each concentration in duplicate, prepared and identified with labels 15 to 105.

The theoretical concentration is the concentration of tolbutamide added to the calibrators.

Peak Area is the integrated area under the peak of interest.

I. S. Area is the integrated area under the internal standard peak.

Ratio Observed is the ratio of the Peak Area to the I. S. Area.

Calculated Concentration is the concentration of each calibrator back-calculated from the curve.

Residual is the percentage difference between the calculated concentration and the theoretical concentration.

The C. V. is the coefficient of variation of the two calculated concentrations.

It can be seen from the above results that the extraction procedure gives an accurate measure of tolbutamide concentration in the test sample over a broad concentration range. This therefore demonstrates the usefulness of the invention in determining concentrations of organic molecular species in test solution samples, where normally they are present in concentrations too small for use with conventional quantitative sampling techniques.

The above results tabulated in Table 1 were plotted on a graph, with peak area versus the concentration. This plot is shown in Figure 3 of the accompanying drawings. It can be seen that the invention produces good curve linearity, demonstrating the accuracy and practical usefulness of the invention over a wide range of concentrations of species to be extracted.

Example 2.-Oualitv control standard results.

To further confirm the above results in Example 1, a second series of experiments was run in the same manner as those of the first series described above, except here each different theoretical concentration was the subject of six runs. Each sample was prepared for a separate weighing of tolbutamide and prepared by a different person and analysed in an identical manner to the calibration curve of Fig. 2 (from Table 1).

The results are shown in Table 2 below.

Table 2

Sample Expected Run Peak IS Area Calculated Status Concentration Area QA1 5. 16 469 13247 5. 77 ngml-l OK QA2 5. 16 359 11771 4. 78 ngml-1 OK QA3 5. 16 235 6784 5. 62 ngml-1 OK QA4 5. 16 384 13651 4. 31 ngml-1 OK QA5 5. 16 470 13555 5. 62 ngml-l OK QA6 5. 16 429 13611 4. 99 ngml-1 OK QB1 70. 67 3298 8132 80. 23 ngml-1 OK QB2 70. 67 3643 10009 71. 86 ngml-1 OK QB3 70. 67 4451 12228 71. 87 ngml-1 OK QB4 70. 67 6169 21032 57. 65 ngml-1 LOW QB5 70. 67 4878 14379 66. 89 ngml-1 OK QB6 70. 67 4116 11526 70.

QCl 203.85 12543 13296 188. 41 ngml-1 OK QC2 203. 85 5638 5770 195. 50 ngml-l OK QC3 203. 85 11904 12434 191. 23 ngml-1 OK QC4 203. 85 12015 12277 195. 51 ngml-l OK QC5 203. 85 8064 8545 188. 98 ngml-1 OK QC6 203. 85 14126 11910 237. 23 ngml-1 HIGH QD1 396. 83 25907 15709 330. 39 ngml-1 LOW QD2 396. 83 30743 17876 344. 60 ngml-l OK QD3 396. 83 19741 10262 385. 61 ngml-1 OK QD4 396. 83 28233 17705 319. 42 ngml-1 LOW QD5 396. 83 20516 10382 396. 16 ngml-1 OK QD6 396. 83 lS709 9252 340. 19 ngml-1 OK In the above Table 2 the column headings have the following meanings: Sample: There are 24 quality control standards, four different concentrations with 6 replicates at each concentration, prepared and identified with labels QA1-6, QB1-6, QC1-6, QD1-6.

The expected concentration is the concentration of tolbutamide added to the calibrators.

Peak Area is the integrated area under the peak of interest.

I. S. Area is the integrated area under the internal standard peak.

Calculated Concentration is the concentration of each calibrator back-calculated from the curve.

Status-The US Food and Drug Administration Guidelines propose that the quality control standards QA1-6 (i. e. at the lowest concentration) must be within +/-20% of their expected concentration to be acceptable. We define these as OK if the results are within these limits. The quality control standards QB1-6, QC1-6, QD1-6, at the higher concentrations must be within +/-15% of their expected concentration to be acceptable. We define these as OK if the results are within these limits. The results given above would conform to the acceptance criteria for a single validation day outlined in the FDA Guideline document"Analytical methods Validation: Bioavailability, Bioequivalence and Pharmacokinetic Studies", Shah, P. V. et al, Pharmaceutical Research, vol. 9, No. 4,1992, page 588.

The above results in Table 2 illustrate the accuracy and consequential practical usefulness of the sampling apparatus and method according to the invention.

Claims (5)

1. CLAIMS 1. An apparatus for sampling one or more species of organic molecules from a test solution, the apparatus comprising: (i) a first solution reservoir for containing said test solution and including a window therein sealed by a non-porous silicone rubber membrane; and (^ii) a second solution reservoir for containing a receptor solution and containing at least that portion of the first solution reservoir which includes the sealed window.
2. 2. An apparatus according to claim 1, wherein the nonporous silicone rubber membrane is of dimethylpolysiloxane.
3. 3. An apparatus for use in sampling one or more species of organic molecules from a test solution, the apparatus comprising a test solution reservoir including a window therein sealed by a non-porous silicone rubber membrane.
4. 4. A method for sampling one or more species of organic molecules from a test solution containing same, comprising: (i) providing an apparatus according to claim 1; (ii) placing the test solution in the first solution reservoir; (iii) placing the receptor solution in the second solution reservoir; (iv) inserting the first solution reservoir into the second solution reservoir such that the latter contains at least that portion of the first solution reservoir which includes the sealed window and such that the sealed window is immersed in the test solution in the second solution reservoir; (v) optionally, allowing the apparatus to stand for a predetermined period of time; and (-vi) removing at least a sample of the receptor solution from the first solution reservoir for subsequent analysis for the desired organic molecular species.
5. 5. An apparatus for sampling one or more species of organic molecules, or a method therefor, substantially as described herein with reference to the accompanying drawings.