CA1084395A

CA1084395A - Method for sampling and fixing urinary metabolites on an immobilizing support and apparatus therefor

Info

Publication number: CA1084395A
Application number: CA277,212A
Authority: CA
Inventors: Jean-Louis Magadur; Georges Morel; Philippe Gauntley
Original assignee: Institut National de Recherche et de Securite INRS
Current assignee: Institut National de Recherche et de Securite INRS
Priority date: 1976-04-29
Filing date: 1977-04-28
Publication date: 1980-08-26
Also published as: US4159193A; DE2719286A1; GB1553455A; SE7704921L; DE2719286C2; JPS52148196A; FR2385377A2; FR2385377B2

Abstract

ABSTRACT OF THE DISCLOSURE

A method for sampling and fixing urinary metabolites on an immobilizing support which is a paper comprising a substance appropriately activated before use. The determination of the or each required metabolite is expressed in relation to the determination of a metabolite where excretion is constant with time. The appropriately activated substance may be an ion exchange resin or silica gel. The method of the invention may be used for determining delta-aminolevulinic acid (ALA), or trichloroacetic acid and trichioroethanol conjugated with glucuronic acid, in urine samples.

Description

~4~95 BAC~GROUNI~ OF TIIE INVE:NTION
The present invention rela-tes to a method for sampliny and fixing ur:Lnary metabolites on an immobilizing support and apparatus therefor.
The present invention enables certain medical analyses to be carried out on large population groups under satisfactory conditions.
It is well known that mass medical examinations~ e.g. in a factory when checking on occupational diseases, encounter numerous difficulties, such as carrying out the biological sampling, particularly of urinel storing the samples, transporting the samples to the laboratory where the relatively : complex analyses can be carried out and the mass arrival ofthese samples at the laboratory, which cannot process all the samples immediately so that some of them must be left waiting.
PRIOR ART
To overcome these disadvantages, and in the particular case of the determination of delta-aminolevulinic acid, a characteristic.metabolite oflead poisoning, Lester ~lankin and Associates (Clini.cal Pediatrics, December 1970, Volume 9, No 13, pages 707 to 712) have proposed that the urine samples should not be transported to the laboratory but that instead the urinary metabolites should be adsorbed at the actual collecting site, on a piece oE paper impregnated with a cation exchanger resin (~o SA 2, sulphonic acid resin in the Na form), by brlefly dipping the paper into the urine, allowing it to dry, packing it suitably and sending it to the laboratory by . post. On receipt of the piece of paper, the labora-tory desorbs the metabolites fixed on the cation exchanger resin by suhjecting it to the action of an acetate buffer and carries out the colorimetric reaction which enables the determination to be made~
.,'' '~ ~
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3~5 ~, t This reactlon comprises forming a pyrrole by reacting clelta-aminolevullnlc acld (AL~) with acetyl acetone, and -then reacting the pyrrole wi-th ~hrlich's reagent, to give a coloured complex whlch ca:n be determined by colorimetry. A
callbration curve prepared from a urea solution is used for the reading.
As far as Applicants are aware, this prior art process has not been used on a large scale and this may be due to the fact -that the colorimetric reaction used for the determination is not specific to the pyrrole formed in the reaction between A1A and acetyl acetone. It is influenced by numerous factors, more particularly the possible presence .; of Ehrlich positive and Ehrlich negative compounds, urea, . etc. in the urine.
Although the Hankin process allows for the presence of . urea by proposing that -the readings should be taken on a calibration curve from a urea solution, it does not allow for the unwanted metabolites comprising the Ehrlich positive and Ehrlich negative compounds. The essential improvement -: 20 to be made to the Hankin process is therefore~ primarily, to , eliminate these unwanted metabolites which falsify the . determination and the simplest solution would appear to be carefully to wash the resin-impregnated paper before desorption of the ALA.' Unfortunately, the paper SA 2 in the Na form used by Hankin does not f.ix -the ALA sufficiently to allow such . washing since the washing results in a loss of ALA which - may be as much as 5.l~ and which,fur-thermore, is not even constant.
It is an object of the presen-t invention to overcome , this disadvantage by proposing an immobilizing support, the i properties of which are such as to guarantee that the wanted : 3.

.

metclbol.ites are suf.ficiently fixed so that the eliminati~
of the unwanted metabolites entails only a small and cor,stant loss of the wanted metabolltes.
SUMMARY OF T~IE INVE:NTION
: 5 The present invention provides a method for sampling and fixing urinary metaboli-tes on an ln~nobili.zing suppor-t ~ which is readily transportable to an analysis centre for deter-- minin~, at least one of the metabolites, which comprises the steps of:
- 10 (i) contacting the support with -the urine so as tosample a specimen thereof and to fix urinary metabolites on ~ . the support;
- (ii) transporting the support to an ana].ysis centre;
.: (iii) removing Erom the support unwanted metabolites , . .
which are likely to distrub the determination of the or each . required metabolite:; and .~. (iv) eluting and determining the or each required , . .
metabolite.;
- . the improvement comprising using as the support a paper comprisin~ a substc~nce appropriately activated beFcjre ~se . and expressing the determination of the or each required `.:
metabolite in relation to the determination of a metabolite whose excretion is constant with time.
.
BRIEF DESCR:[PTION OF TIE~DR~WINGS
In the drawings:
; Figu:re 1 is a graph of ALA scales produced from the - results o:E measurement on ten u.rine samples usincJ Mauzerc -, and Gran:lck's technique;
'~ . Figure 2 is a graph o AI.A scal.es produced frorn the -~ 30 results of measurement on t:en uri.ne samples using Grabeclci's . -s: techniqlle;
~ . .... ~?igures 3 and 4 il~ust:rate; an apparatus ~.or use in the .: '1.
;

3g5 method hf the invelltion;
Fiqure 5 is a calibration curve ~or ALA values obtained accorclin~ to -the method of the invention; and Figure 6 is a ca]ibration curve for creatinine values obtained according to the rnethod of the invention.
DESCRIP'rION_OF T~ PREFERRE:D ~3MBODIMENTS
More specifically, in carrying out the method of the invention, the support used is preferably a paper compri-sing a material which is suitably activated before use.
The paper treated with the suitably activated material itself forms the sàmpling rneans and it will be understood that the amount of the sample depends on the quality of the paper and that for a paper of constant quality the amount of urine sampled can be known with good accuracy.
Nevertheless, to avoid the results being falsified by variations in the level of the amount of urine sampled or, in particular, by variations in the dilution of the urine - which varies greatly according to the time at which the sL~mpling i5 made and the amount of liquid intake, etc. the result of the determi.nation of the or each wanted metabolite is expressed, in the method of the invention, relative to the result of the determination of a metabolite whose ' excretion is constant per unit of time.
-!
In a first embodiment oE the invention, the suitably activated material comprised by the ~aper is an ion~exchange ~ resin.
~,:
- The resins suitable for carrying out the method of the invention are selected, for example, from those whose properties are glven in the following Table I, although i-t is to be understood that -this list is not limiting.

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TA~3LE I

. . ....... . . . . . ._ _ __ _ Skeleton Eunctional ~roup Functional type - . . . ___ Polystyrene ArS03H strong acid Aliphatic polymer -COOH weak acid Polystyrene -NtCH3)2 strong base Copolymer Pol~amine weak base Taking the specific case of the polystyrene skeleton : resin with a functional group ArS03H (which corresponds -to :- the form Na resin used to impregnate the No SA 2 paper used by Hankin), activation comprises transferring it to a more acid form chosen between NH4 , Li and H . To achieve this result, the ion-exchange resin is contacted with a compound adapted to provide it with the required ion, such as NH40H, LiCl or HCl.
In the activated H form, the polystyrene skeleton resin of functional group ArS03H fixes ALA much better than in the ~ original Na form. This is apparent from the tests shown in - Table II below, in which strips of No SA 2 paper, H activated and Na non-activated, were impregnated with 100 ~1 of a 40 mg/l ALA solution with an addition of 1.6 mg/l of highly -: radioactive ALA (125600 disin-tegrations per minute), and the .: impregnated strips were washed twice.
~TABLE_II
_ ~ . , ALA fixed Loss on first Loss on second Total _ _ wash wash loss H 169 ~ 9.3 1 ~ 0.1 0.37 + 0.02 1.38 + 0.03 Na .ll5 + 9.~ 29 + 3 16 -~ 1.4 45 + 4.3 .~
Tests have shown that for carrying ou-t the method of the invention Eor de-termining -the ALA i-t is preferable to activate the polys-tyrene skeletonlresin of -the func-tional group ArS03H

.~ 6.

3~i so that it ls in the ll fornl rather than in another activated form, and the results set out ln Table III show that the N~14 and Li forms retain even less ALA than the non-ac-tivated Na f orm .
TABLE III
' . __, ._ . . . _ . _.
Strips of paper Activated Eorms Non activated form impregnated with _ Na polystyrene ~ + +
ArS03H resin NH~ Li H
. . _ . . .. . . ~
Quantiky fixed 100% lC0% 100~ 100~
Loss on lst wash 83% + 10 76~ ~11 1% ~ 0.1 30% + 4 Loss on 2nd wash 12% ~ 2 15% + 2 0.4% + 0.0421~ + 3 Recovered from the eluate 5% + 0.6 9~ + 2 98% + 11 49% _ 5 The unwanted metabolites fixed on the activated resin - are eliminated by repeated washing in water with agitation - (for example twice for 10 minutes), whereupon the resin is dried.
he ALA is then desorbed by bringing the resin into contact with an acid buffer and the ALA is determined by the general use of the coloured reaction obtained with Ehrlich's - reagent under the conditions set out hereinbefore.
Various urinary ALA determination techniques have also been previously proposed.~ They are techniques related to those of Mauzerall and Granick and to that of Grabecki.
A - Mauzerall and Granick related techniq~es - Urine samples are passed through two ion-exchange columns, one an anion and the o-ther a cation exchange column, so that the endogonous pyrroles and Ehrlich positive substances are eliminated, the urea is eliminated and certain Ehrlich nega-tive substances which have an interfering ac-tion are removed. The final dilution is 1/28. A s-tandard reading 7~

, ~ . . . . . .. . .. .

3~35 curve is prepared in water without passage through the resins. ALA scales produced by this -technique from 10 urine samples ~ive a dispersion of -the resul-ts as shown in ~igure 1 of the accompany:Lng drawings. The fact that the stan~ard curve prepared in water is used results in some uncertainty due to syst:ema-tic default and this may be as much as -38% of the true val~le.
B_- Grabecki related techniques These techniques are characterised by the fact that the Ehrlich negative substances are not extracted. The contribution of the Ehrlich posi-tive substances is deduced by measuring a "blank urine". The final value is 1/14.
I The reading may be made in two ways:
a) On a standard_curve corresponding to a scale in water Figure 2 of the accompanying drawings shows the dispersion obtained against the urines. The uncertainty in respect of determination is systematic and by default. It may be as much as -65~. The technique studied has a final dilution of 1/20.
b) By means of an internal standard . In this case, the reading i5 carxied out on a curve :~ prepared from the urine under analysis. There is no longer any dispersion due to the urines. Random uncertainty of determination persists, hbwever, and may be as much as + 5%.
In respect of the determination of ALA, only one technique gives satisfactory results which is that derived from the Grabecki method, using an internal standard. However, this ~ technique is applied directly to the urine and not to an `- eluate of urinary metabolites. The present Applicants have therefore adapted this technique to the specific case of the determination of ALA desorbed from an activated resin and in their me-thod the optical density of the coloured comple~

: 8.

. ~` ' .

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prcduced by Ehrlich's reaction and recti~ied by comparison with the optical density of a blank which has received only Ehrlich's reagent and which has not received acetyl acetone is read on a calibration curve for ALA in water and S determined by means of the activated resin.
The correction carried out on the basis of the optical density of the blank makes it possible to allow for the reactions of Ehrlich's reagent with compounds other than pyrrole formed from AL~ and which are not eliminated on washing.
Also, the use of a standard scale prepared in water and determined by means of the activated resin eliminates the risk of error due to the latter. The accuracy of determinatiGn is + 5%. ~;
- 15 In another aspect of the present invention, the suitably acti~ated material c~mprised by the paper is a silica gel.
~ ., A paper of this kind may be used, for example, for determining trichloroacetic acid and trichloroethanol conjugated with glucuronic acid, such urinary metabolites being indicative of trichloroethylene poisoning.
The choice of this type of immobilizing paper for carrying out the above determination was made by subjecting various commercial immobilizing papers to fixing tests using tri-chloroacetic acid labelled with carbon 1~
The performances of the silica gel treated paper will be ~ -.
apparent from Table IV which indicates the following:-the nature of the solution for washing the strip of paper after immersion in the solution containing the labelled trichloroacetic acid, and dry:ing, the percentage loss of labelled trichloroacetic acid in each wash in comparison with the quantity retained; ~ ~
`-~--~the nature of the washing-out solution; and ~ ~ -.. =, . . , . ~ . .. . .

1 ~843gS

the percentage of trlchloroacetic acid finally washed out by comparlson with the quantity retained.
TABLE IV
Silica gel -treated paper Wash solution Benzene Cyclohexane Percentage of lst wash 2~ 0 TCA loss relatively to quantity retained 2nd wash 1.7~ o 3rd wash 1~ o Washlng-out solution Water or Phosphate phosphate buffer buffer ; Percentage of TCA
- washed out relatively to quantity retained 93~ 98%
TCA = trichloroacetic acid It will be noted that washing is not carried out with water, but with a suitable non-polar solvent. Cyclohexane gives satisfactory results. Prior to use, the silica gel trea-ted paper is activa-ted by dehydra-tion. The amounts of : i ` trichloroacetic acid and trichloroethanol in the eluate are : ~ , ~ determined by means of the method of Ogata et al.
.
~- In a first stage -this method comprises hydrolyzing the conjugated trichloroe-thanol and then, in a second stage, reacting the trichloroacetic acid and the Liberated tri-chloroethanol with pyridine in a hot alkaline medium. A
reddish-orange colour develops. By means o~ calibration curves, the trichloroacetic acid and trichloroethanol concentrations can be read off from the co:Lorimeter at two different wavelengths.

In the method of the present invention, urinary creatinine can be determined using paper treated with an ion-exchange resin or silica gel.
It is well known that the determination of creatinine ~-the elimination of which is substantially constant through-~' . :LO.
~
~ ~ .
. ~ , .

3~i out the day) may be used to determine the dilution of khe urine sample and to rectify the results of o-ther tests carried out on the urine sample depending on such dilution.
More particularly, it is conventional to express the de-termination of ALA in -terms of mg of ALA per g of creatinine (standardization of the result).
In the presen-t method, this standardization technique also offers the advantage of eliminating any risks of error due to the use of an immobili~ing paper as the sampling means and hence due to the absence of an exact measurement of the amount of -the sample taken.
The method of the present inven-tion has the advantage - of enabling the or each required urinary metaboli-te ~e.g.
ALA or conjugated trichloroethanol and trichloroac0tic acid) - 15 and the urinary creatinin~ to be sampled in a single operation using the same immobilizing suppor-t.
The activated material on which the wanted urinary .
metabolites and -the creatinine are both fixed, is washed, dried, and washed out, as described above. An aliquot part of the eluate is then used for determination of the or each wanted urinary metabolite whilst another aliquot part is ; used for determination of the creatinine. The result Eound for the or each wanted metabolite is -then related to that -~ found for the creatinine.
As for the determination of the ALA, a polystyrene skele-ton resin having a functional group ArS03H activated so as to be in the H form, which is more satisfactory than the NH~ and Li forms, may be used for sampling and fixing the creatinine.
This will be apparent from Table V:

11 .

.~ .

~43~i .. ~
TAsI~E V
~ . . .__ . _ Strips of ~aper impr~lnated Activated forms Non-activated forms with polystyrene ArS03 4 Li H Na ~ .. .... .. .__ ~ ... ~
Quantity fixed: l00% 100% 100% 100~
- Loss on 1st wash 56.55 48.4 4.8 68.5%
Loss on 2nd wash 16.5 15 3.8 16%
Recovered from the eluate 26.8 36 91.4 15.4%
It will be seen from this Table -that the activated form Li+ retains creatlnine -the compound be-tter than the non-` activated Na~ form, although this is not the case with the ALA. The activated resin on which the crea-tinine i5 fixed is treated as hereinbefore described with reference to the ALA determination. The resin is washed several times with - 15 water, with agitation, dried, and then desorbed by bringing it into contact with an acid buffer solution.
The creatinine is determined by Jaffe's method in which the creatinine is reacted in a basic aqueous medium with a ., sodium picrate solution to form a coloured complex which is determined colorimetrically. In -the specific case of the determination of creatinine washed out from a resin, the effect of the acid buffer used for the desorption of the creatinine must be neu-tralised prior to, or simultaneously with, the in-troduction>of the reagen-ts required for Jaffe's - 25 reaction. If this is not done, the amount of base to be added according to Jaffe's reaction will not be sufficient :: .
~ to bring the medium to a basic pH and the reaction will not :';
take place.
As for the determination of ALA, the optical density of Jaffe's reaction proAuct is corrected by allowing for the ~; optical densi-ty of a blank in which the amount of eluate has been replaced by a corjresponAing amount of water, and the 12.
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3~i value fo~nd is read ofE from a cal:ibration curve in respect of creat:inine in water determined by means of -the activated resln.
A sllica qel treated paper may be used for sampling and fixing the creat:Lnine. The creatinine is then washed ou-t as clescribed in connec-tion with the determination of the trichloroacetic acid and trichloroethanol~ and determined by Jaffe's method as described above.
The present inven~ion also rela-tes to apparatus for carrying out the method of the inven-tion. This apparatus comprises:
a tube closed a-t one end and open at the other end;
a plug adapted to provide a sealing closure of the open end of the tube;
a paper strip treated with an appropriately activated substance and received in the tube; and : means for securing the paper strip to the plug.
In a preferred embodiment, the tube comprises a stop : member adapted to immobilize a body of a dehydratiny product against the tube end, e.g. a silica gel pellet.
Advantageously, the plug is hollow and the strip-securing means is an elastic member received in the p~ug recess or cavity, such member being provided with a slot in the plane of the longitndinal axis of the tube, -the slot substantially separating the member into two parts inter-connected only by a bridge or web of material and being . adapted to be opened to receive one end of the paper strip,and closed to immobilize -the strip when the elastic member is introcluced into the plug recess or cavity.
In order to increase the area of contact between the elastic member and the s-trip, the slot is preEerably bounded by solid walls. These walls advan-tageously have projections 3g5 which contribute to immobilizing the strip.
Two embodlments of the method of the present invention will now be described by ~ay of specific example.
_~MPLE 1 Early cliasLnosis of_lead poisonin~
The invention ls described in this Example with reference to Figures 3 and 4 oE the accompanying drawing, which are respectively a perspective view in exploded form before assembly and a section after assembly of one embodimant o~ the apparatus of the present invention.
Referring to Figures 3 and 4, the apparatus comprises a tube 1, closed at one end by an end 2 and open at the other end 3. The open end 3 can be closed so as to be sealed by a hollow plug 4, provided with a gripper tab 5. An elastic member 7 subdivided into two compartments 8a and 8b fits into the cavity 6 of the plug ~. The two compartmebts 8a and 8b - are connected only by a bridge of material 9 and together define a slot 10. Since the material of member 7 is elastic, the edges of the slot 10 can be moved apart and a strip o~
paper 11 inserted therein. Once the strip 11 i5 in position in the slot 10, the member 7 is inserted into the cavity 6 of the plug 4, where it is immobilized by its projecting periphery 12 bearing on a corresponding shoulder 13 provided - on -the inner surface of~-the plug 4. It will be seen that in ; 25 this way the strip 11 is connected -to the plug 4. The strip 11 consists, ~or example, of paper SA2, i.e. paper impregnated wlth a polystyrene skeleton resin having activated ArS03H
functional groups.
To actlvate the resin, the s-trip impregnated with the non-activated resin is immersed for 15 minutes in a 2 N hydro-; chloric solution (bi distilled water) which is periodically agita-ted. Bi-distilled wa-ter is -then used for washing until 14.
:.
.
.:
, -8'~395 the lnitlal p~l of the bi-distilled water ls restored, and dryiny is carried out in atmosphere screened ~rom light and basic fumes.
The strip 11 has a reference such as a mark (not shown) where it will be subsequently cut. The reference obviously enables the same strip length always to be cut so that the same amount of resin is always used. A dehydrating pellet 1~ (formed, for example, by activa-ted silica gel ~ontaining a coloured saturation indicator) is slid inside -the tube.
This pellet is held in place by a stop member 15 which is force-fitted in the tube. Before use, the appara-tus is in the form shown in Figure ~. The apparatus is used as - follows:
Samplin~ '!
The operator who receives the apparatus packed in opaque packing removes it therefrom and then removes plug ~
from tube 1 holding it by gripper tab 5. The strip of paper is then removed from the tube 1. The strip is immersed in the urine to be tested for about one second, wiped along the wall of the container containing the urine, and replaced in the tube by re-inserting the plug. During -these operations it is aclvantageous to avoid exposure to bright light (direct sunlight, or a nearby fluorescent tube) and the presence of ammoniacal fumes in -the~surrounding atmosphere, since such fumes may affect fixation or the storaye oE the substances under analysis. ~he complete device is then placed in an .:;
;; envelope designed for this purpose and suitably identified, ~ e.g. of the kind used for the dispa-tch of cine films to:
processing laboratories, and posted.
All of these opera-tions are simple and fast to carry out and no skilled personnel is re~uired. Conse~uently, this -type of sampling is very suitable for mass examination, 15.
'''' ~ 8~3~5 e.g. examination in a factory. After sampling, the strip i5 kept in the tube, where it finally drles by the dehydratin~ action of pellet 1~. The strip can be left in this way for several days without harm, and this solves the problem of mass and interm:it-tent arrival of samples at the laboratory.
Treatment at the labo~ y For analysis, the tube is opened, the strlp is cut a-t the reference mark (the remainder being kept for control lQ purposes, if necessary) and -the cu-t portion of the strlp is washed with distilled water by placing the piece of the strip in a dish containing 5 ml of bi-distilled water, the dish being positioned on a plate receiving a gentle and irregular movement. After immersion for 10 minutes the bi-distilled water in the dish is replaced by a fresh quantity of 5 ml of bi-distilled water and the immersion operation is repeated, with agitation, for 10 minutes. These prolonged and repeated washes eliminate most of the substances which are likely to interEere with subsequent analysis. The piece of strip is dried in the air and then immersed for 10 minutes, away from light, in 5 ml of 1 M acetate buffer p~l ~.6. The metabolites fixed on the resin are desorbed and the eluate from this treatment is used for -the determination of the ALA
and creatinine. '~
ALA determination ..... _ .
25 ~1 of acetyl acetone are introduced into a first tube and heated for 20 minutes on a water bath at 100C to form a ; pyrrole. The contents of -the tube are allo~ed to return to ambient temperature whereafter 1 ml of Ehrlich's reagent is added to give a characteristic red colouring. The same operation is repeated in a second tube in which the amount of ace-tyl acetone is replaced by a corresponding amount oE

16.

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101~395 water. This tube acts as a ~lank f~r possi~le correction of the colouring given by ~hrlich's reagent ~ith substances other than the pyrrole frorn t}le AL~. With a wavelength of 546.10 9m, the colorimetre is set to O OIl the blank (second - 5 tube), anci then the optical density of the contents of the first tube is measured beincJ, for example, 0.268. To allo~
for the risk of error due to the resin and the paper of the strip, a correction is carried ou-t by reading the above result on a water calibration curve prepared as follows: a range of solutions of ALA in water is prepared wit~
increasing concen-trations (from 2 mg by 2 mg steps per 100 ml) and sampling is carried out by means of strips as if urine were involved. The strips then receive the same treatments -- as indicated above, i.e. washing, desorption, Ehrlich's `~ ~15 reaction and measurement of the optical density for each ALA
' concentration relative to a hlank. The values found enable . .
the curve shown in Figure 5 to be plotted. The optical density found for the sample, i.e. 0.268, corresponds to : . , - an ALA content of 20 mg ALA/l according to the water calibration curve.
Determination of creatinine ; Three drops of 20% NaOH (20 g/100 ml) and 1 ml of eluate are introduced into a first Eppendorf tube and agitated for 30 seconds to neutralize the acetate buffer used for the ., ,j , .
desorption. 200 ~1 of saturated picrate acid and then 100 of 7~ NaOH are sampled. ~gitation is carried out for 30 , . .
- seconds to obtain thorough mixing and the mixture is left for 20 mlnutes in clarkness during wllic}l time an orange colour .: `
forms. ~ blan}c is prepared in a second Eppenclorf tube using ,~ 30 t:he above procedure but replacing the 200 ~1 of eluate witll .',:! , 200~1 of distilled water. With a wavelen~Jth o 546 nm Lhe ~ ~~colorimetre is set to 0~ on the b]ank (second tube) and then ; ~7.

.

:~ -3~5 the optlcal density of the contents of the first -tube is measured being, for example, 0.459. As in the case of the ALA determina-tion, to allow for the risk of error due to the res.in and the paper of the strip, a correction is carried ou-t by reading the above result off Erom a water calibration curve prepared in the same way as the ALA
calibrati.on curve, and which is shown in Figure 6. The optlcal density measured above corresponds t.o a creatinine content of 2 g/l. Comparison of the resul-ts (20 mg ALA/l and 2 g creatinine/l) shows that the urine of the subject contains 10 mg ALA/g of creatinine.

Earl~agnosis of trichloroeth~ ene poison_ng In this case, a silica gel treated paper was used : 15 packed in the same way as the ion-exchange resin treated paper described in Example 1. Activation of the silica gel was effected by a dehydrating pellet 14 provided to dry the : strip of paper after immersion in the urine and draining.
The urine is sampled in the same way as the sampling . 20 carried out for the determination of ALA.
Treatment in the laborator~
After opening the tube in which the strip is replaced after sampling, the strip is cut at the reference mark and the cut part washed.
The procedure of Example 1 is followed using cyclo-he~ane instead of water. Unwanted metabolites are removed in this way and the strip of paper is dried in air and then immersed for 10 minutes in 5 ml of wa-ter or 0.2 M phosphate buffer ~pH 6). This operation liberates the metabolites fi~ed by the silica.
The eluate obtained from the above treatment is used -to determine the amounts of -trichl.oroacetic acid, -trichloro-:L8.

:
,:,.

3~5 ethanol and creatlnine.
Determination of trichloroacetic acid and trichloroethanol . _ . . _ _ . .
200 ~1 of eluate and 10 ~Il of ~-glucuronidase ~750 Fishman units) are in-troduced into a tube which is then heated at 37C for 40 minutes to hydrolyze the conjugated trichloroethanol, whereafter 200~1 of 50~ NaO~I and 1 ml of pyridine are added to the tube and heated at 95C for 5 minutes. A reddish-orange colour develops. ~fter cooling, 800~1 of the pyridine layer is sampled and 200~1 of water added thereto. Two readings are taken with -the colorimetre, one at 436.10 m to measure the trichloroethanol concen-tration and the other at 546.10 9m to measure the trichloroacetic acid concentration and the results are obtained from calibration curves.
To prepare the calibration curves, a range of solutions is prepared which respectively contain 100, 300, 500 and 1000 mg/l of trichloroacetic acid and trichloroethanol (it would of course be more exact to start with conjugated tri-i chloroethanol, but this compound is not available in this -- 20 form and free trichloroethanol must therefore be used).
To plot the calibration curve for trichloroacetic acid, a sample is taken of each of the solutions of the range by using an identical strip -to -that used for sampling the urinQ
and this strip is treated3in the same way.
The trichloroethanol calibration curve is prepared similarly but by dilu-ting beforehand solutions -to 100, 300, 500 and 1000 mg/l and samplin~ the solu-tions using the same paper strip.
From these two calibra-tion curves it is possible to - 30 determine that a urine sample treated by way of experiment and found to have an op-tical density oE 0.280 at 436.10 9m, has a trichloroethanol cloncentration of 500 mg/l, and a L9.
., :`:
.~,.................... : . .
:-"': . ' ' , ' sample ound to have an optical density of 0.400 at 5~6.10 9m has a trichloroace-tic acid concentration of 300 mg/l.
Determina-tion of creatininl~
The determination was carried out as in Example 1.
200~1 of eluate, 100 ~1 vf 7% NaO~I and 500 ~1 of saturated picric acid in water are in-troduced into a first tube, a~itated for 30 seconds, and then leEt to stand for 20 minutes. An orange colour develops. A reactive blank is prapared in a second tube. The optical density is measured at 546.10 9m by setting -the colorimetre to O for .
the blank.
With the same urine sample as used for determination of the trichloroacetic acid and -trichloroethanol an optical density of 0.410 is found, and reference to a previously plotted calibration curve shows that this corresponds to a concentration of 2 g/l.
: Standardization of results : --------- . . . _ c Comparison of the results (500 mg/l of trichloroethanol . 20 and 300 mg/l of trichloroacetic acid/2 g/l of creatinine) ;~ shows that the urine of the subject contains 250 mg of tri-~ chloroethanol/g of creatinine and 150 g of trichloroacetic ; acid/g of creatinina.
. From the foregoing description it will be appreciated - 25 that the method of the present invention enables a considerable number of samples to be processad under the : most satis:Eactory conditions (storage, accuracy of result, ~:~ cost-price etc.).
', , . . .

, 20.
:
,.~

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

l. In a method for sampling and fixing urinary metabolites on an immobilising support which is readily transportable to an analysis centre for determining ------at least one of the metabolites, which comprises the steps of:
(i) contacting the support with the urine so as to sample a specimen thereof and to fix urinary metabolites on the support;
(ii) transporting the support to an analysis centre;
(iii) removing from the support unwanted metabolites which are likely to disturb the determination of the or each required metabolite; and (iv) eluting and determining the or each required metabolite, the improvement which comprises using as the support a paper comprising a substance-------appropriately activated before use and expressing the determination of the or each required metabolite in relation to the determination of a metabolite whose excretion is constant with time.
2. Method according to claim 1 wherein the support is in the form of a narrow paper strip which is dipped into the urine to be sampled.
3, Method according to claim 1 wherein the appropriately activated substance is an ion exchange resin.
4. Method according to claim 3 wherein the resin is selected from the group consisting of rosins having a polystyrene skeleton and an ArSO3H fuctional group, resins having an aliphatic polymer skeleton and a -COOH functional 21.

group, resins having a polystyrene skeleton and a -N(CH3)2 functional group and resins having a copolymer skeleton and a polyamine functional group.
5. Method according to claim 3 wherein the resin is a resin having a polystyrene skeleton and an ArSO3H functional group, the resin being activated by being transferred from the Na+ form to a form selected from the group consisting of the NH4+ form and H+ form.
6. Method according to claim 3 wherein the resin is activated by being contacted with a compound which provides it with the required ion.
7. Method according to claim 1 for determining delta-aminolevulinic acid (ALA) wherein a urine sample is taken by means of a paper strip containing a polystyrene skeleton resin having an ArSO3H functional group activated in the H+
form, the strip is dried, washed to remove unwanted metabolites, the washed strip is dried and washed out, and the ALA is condensed with acetyl acetone to form a pyrrole to which Ehrlich's reagent is added -to form a stained complex which is determined colorimetrically.
8. Method according to claim 7 wherein the optical density of the pyrrole complex, rectified by comparison with the optical. density of a blank which has received only the Ehrlich's reagent and which has not received acetyl acetone, is read on a calibration curve.

22.
9. Method according to claim 7, wherein the colorimetric measurement is made at a wavelength of 546 10-9m.
10. Method according to claim 1 for determining creatinine wherein a urine sample is taken by means of a paper strip containing a polystyrene skeleton resin having an ArSO3H functional group activated in the H+ form, the strip is dried, washed to remove unwanted metabolites, the washed strip is dried and washed out and the creatinine reacted in a basic aqueous medium with a sodium picrate solution to form a coloured compound which is determined colorimetrically.
11. Method according to claim 10 wherein the optical density of the coloured compound, rectified by comparison with the optical density of a blank for which the test sample containing the creatinine has been replaced by a corresponding quantity of water is read on a calibration curve.
12. Method according to claim 10 wherein the colorimetric measurement is made at a wavelength of 546.10-9m.
13. Method according to claim 1 wherein the appropriately activated substance is a silica gel.
14. Method according to claim 13 wherein the silica gel is activated by dehydration.
15. Method according to claim l for determining urinary trichloroacetic acid wherein a urine sample is taken by means of strip of paper containing silica gel, the strip is dried, 23.

washed in an appropriate non-polar solvent to remove unwanted metabolites, the washed strip is dried and washed out and the eluate reacted with pyridine in a hot alkaline medium, to develop a reddish-orange colour which is measured colorimetrically at an appropriate wavelength and compared with a calibration curve.
16. Method according to claim 15 wherein the measurement is made at a wavelength of 546.10-9 m.
17. Method according to claim 1 for determining urinary trichloroethanol conjugated with glucuronic acid wherein a urine sample is taken by means of a silica-gel treated paper strip, the strip is dried, washed in an appropriate non-polar solvent to remove unwanted metabolites, the washed strip is dried and washed out, the trichloroethanol conjugated by .beta.-glucoronidase is hydrolyzed and the hydrolyzed eluate reacted with pyridine in a hot alkaline medium, to develop a colour which is measured colorimetrically at an appropriate wavelength and compared with a calibration curve.
18. Method according to claim 17 wherein the measurement is made at a wavelength of 436.10-9m.
19. Method according to claim 1 for determining trichloroacetic acid and trichloroethanol conjugated with glucuronic acid wherein a urine sample is taken by means of a strip of paper containing silica gel, the strip is dried, washed in an appropriate non-polar solvent to remove unwanted metabolites, the washed strip is dried, washed out, the trichloroethanol conjugated by .beta.-glucuronidase is hydrolqzed 24.

and the hydrolyzed eluate is reacted with pyridine in a hot alkaline medium to develop a reddish-orange colour which is measured colorimetrically at a wavelength of 546.10-9m to determine the trichloroacetic acid and at a wavelength of 436.10-9m to determine the trichloroethanol.
20. Method according to claim 1 for determining creatinine wherein a urine sample is taken by means of a strip of paper containing silica gel, the strip is dried, washed in an appropriate non-polar solvent to remove unwanted metabolites, the washed strip is dried and washed out and the eluate is reacted in a basic aqueous medium with a sodium picrate solution, to develop an orange colouring which is measured colorimetrically at an appropriate wavelength and compared with a calibration curve.
21. Method according to claim 20 wherein the measurement is made at a wavelength of 546010-9m.
22. Method according to claim 1 wherein the result of the determination of the or each required metabolite is expressed relative to the urine creatinine level.
23. Method according to claim 22 wherein the ALA
and the creatinine are determined on two fractions of the same eluate.
24. Method according to claim 22 wherein trichloro-acetic acid and trichloroethanol, on the one hand, and 25.

creatinine, on the other hand, are determined on two fractions of the same eluate.
25. An apparatus for carrying out the method of claim 1 which comprises:
a tube closed at one end and open at the other end;
a plug adapted to provide a sealing closure of the said open end of said tube;
a paper strip treated with an appropriately activated substance and received in said tube; and means for securing said paper strip to said plug.
26. Apparatus according to claim 25 wherein said strip is a paper strip containing ion exchange resin.
27. Apparatus according to claim 25 wherein said strip is a paper strip containing silica gel.
28. Apparatus according to claim 25 wherein said tube includes a stop member adapted to immobilize a body of dehydrating product against the tube end.
29. Apparatus according to claim 25 wherein said body of dehydrating product is an activated silica gel pellet containing a coloured saturation indicator.
30. Apparatus according to claim 25 wherein said plug is hollow and said strip-securing means comprises an elastic member received in the plug recess or cavity, the said elastic member being provided with a slot in the 26.

plane of the longitudinal axis of the tube, the said slot substantially separating said elastic member into two parts interconnected only by a bridge or web of the elastic material, said slot being adapted to be opened to receive one end of the said paper strip and closed to immobilize the said paper strip when said elastic member is introduced into said plug recess or cavity.
31. Apparatus according to claim 30 wherein said slot is bounded by solid walls.
32. Apparatus according to claim 31 wherein said solid walls have projections.
33. Apparatus according to claim 25 wherein said plug has a gripping member.

27.