AnaQtzca Chumca Acta, 275 (1993) 275-278 Elmer Science Pubhshers B V , Amsterdam

275

Extraction and clean-up of the P-agonist salbutamol from liver and its determination by enzyme immunoassay

L Howells, M Sauer, R Sayer and D Clark Central Vetemmy Laboratory New Haw, Weybndge, Surrey KT15 3NB (UK)

(Recewed 20th May 1992, rensed manuscrtpt received 10th December 1992)

Abstrael

A method has been developed for the extractlon and unmunoassay of salbutamol m hver The procedure involves dIgestion of hver urlth Subtdlsm protease at pH 10 5, clar&a~on by acid preapltatlon and centnfugatlon The salbutamol m the supematant was concentrated onto a 2-g Cls “Bond Elut” cartridge at pH 9 1 f 0 3, eluted with 99 5% methanol and the eluate concentrated and quanhtated by enzyme nnmunoassay The method was vahdated usmg tissues m which salbutamol had been incurred and trues sprkcd wtth salbutamol The mean recovery from hver spked at 1 pg kg-’ was 57 f 8% (S D ) Statistical analysti of the results for 18 batches of samples enabled the calculation of the probab&y of false negatives (sensltlvlty) and of false posltnres (spec&‘ic~ty) of the method The probablhty of false negatives among samples contammg 1 pg kg-’ was 1% and that of false positives was 2 5% The method was used successfully on incurred tissues, and IS used routinely m this laboratory for screening samples taken m compliance urlth the requirements of the EC Residues Directive (86/469)

Keyworuk Enzymatic methods, Immunoassay, &Agonists, Lver tissue, Salbutamol

The admnustratlon of high doses of & adrenoceptor agomsts c/3-agonists) such as clen- buterol, terbutahne and salbutamol to food am- mals has been shown to increase carcase lean/fat ratios [l] Although such use 1s lllegal throughout the European Commumty, mcldences of human food poisonmg where &agonists were nnphcated have been reported [2] Consequently, mterest has focused m recent years on the abuse of /3- agonists both m the Umted Kmgdom and m Europe Under the UK “Residues m Meat” pro- gramme, liver samples from randomly selected ammals are momtored for the presence of these compounds

Several workers have devlsed methods for the analysis of salbutamol m plasma by hqmd chro- matography (IX) [3-81, and by gas chromatogra- phy-mass spectrometry (GC-MS) 19-121 but

plasma 1s inappropriate for the surveillance of @agomsts because of the low concentrations present [ 131

Investlgatlon of LC with amperometnc and W absorbance detectors, combmed wth a van- ety of sample clean-up and concentration ap- proaches, faded to show the combmatlon of free- dom from co-extractlves and high recovery which 1s necessary rf residues are to be detected at 1 pg kg-’ m hver (unpublished data) The particular problems found may have been related to partlcu- lar chenucal attributes of salbutamol For exam- ple, because it IS a basic compound It may be subect to protem bmdmg and consequent poor recoveries, especially if protein precipitation techmques are used to prepare extracts [141 Therefore we have investigated the use of a Sub- tlhsm Carlsberg protein digestion step [14] m the

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276 L Howe.% et al /Anal Chrm Acta 275 (1993) 275-278

present study AdditIonally, salbutamol IS charged at all pH values and does not readdy lend itself to nmple, specific back-extractmg procedures This severely restricts the options for sample clean-up

Immunoassay offers a sensitive altematlve to LC for analysis of sample extracts This approach 1s investigated here, using the cross-reaction of a clenbuterol enzyme immunoassay (EIA) to en- able analysis of liver extracts, followmg their con- centration on octadecyl sdane extraction car- tridges

The procedure developed for salbutamol anal- ysis was applied to the analysis of 180 randomly chosen bovme and porcine liver samples

EXPERIMENTAL

Mater&s Salbutamol (free base), Subtlhsm Carlsberg

and p-mtrophenyl phosphate, were obtamed from Sigma Tntmm labelled salbutamol was a gfi from Glaxo (Ware, UK), and tntmm-labelled clenbuterol was a gift from Boehrmger-Ingelhelm (Bracknell, UK)

Alkaline phosphatase (ALPl-12G) was from Blozyme Las (Blaenavon, UK) Ultuna gold XR liquid scmtlllatlon fluid was obtained from Can- berra-Packard (Readmg) The alkaline phos- phatase-clenbuterol conjugate and the clen- buterol antiserum were prepared as descrrbed elsewhere I151

Other chemicals were of analytical reagent grade and were purchased from BDH Merck Salbutamol standard solutions were prepared m water and stored at 4°C The dlethanolamme (DEM) buffer (1 M, pH 9 8) also contamed mag- nesium chloride (0 5 mM) and sodium azlde (0 2 g 1-l) The phosphate salme gelatm buffer (PAS-gelatm) consIsted of 0 1 M phosphate buffer contammg gelatm (1 g l-l), sodmm chlo- ride (9 g l-l), sodmm azlde (0 2 g 1-l) adjusted to pH 7 0 The p-mtrophenyl phosphate (0 02 M in DEM buffer) was prepared 20 mm before use and was kept protected from hght until needed

The LC system consisted of two Pye Umcam Model 4015 pumps with an mterface, a Model

4850 controller and data handlmg system (Un- icam, Cambndge) and a Sevem Analytical 6500 W detector set at 233 nm The heated magnetic stirrer with water bath and thermostatic probe were made by IKA and supplied by Sartonus Instruments

Extractwn procedure Liver samples (10 s> were prepared m batches

of twelve two samples from untreated ammals (controls) were included m each batch, one of which was spiked at 1 pg kg-’ with radlola- belled salbutamol (3500 dpm) Samples were ho- mogemsed m 10 ml Trrs buffer (1 M, pH 10 5) Protease (10 mg), and a further 20 ml of Trls buffer and a magnetic stlrrmg bar were then added to each tube, the tubes were capped and incubated m a water bath on a heated magnetic stirrer for one hour at 55°C Durmg this mcuba- tlon the sample was completely solubdlzed apart from a small amount of connective tissue The protease was subsequently deactivated by lm- mersmg the tubes m bollmg water for 15 mm When cool, the digest was clarfied by acrdlfymg Hnth concentrated hydrochloric acid (3 5 ml) and centrlfugation (30 mm, 1500 g) The supematant was decanted mto a fresh tube and its pH was adjusted to 9 1 with NaOH (10 M) prior to solid phase extraction This pH has been found to be optnnal for the retention of salbutamol on the bond Elut cartridge

Solzd phase extractwn Cl, Bond Elut cartndges (2 g, Analytichem,

Code 1125-6015) were fitted with 75-ml reservoirs contammg a 20-km plastic tit and set up on a “Vat-Elut” box The columns were prewashed with 5 ml of methanol and 10 ml of water The samples were poured mto the reservoir and al- lowed to percolate slowly (not more than 5 ml mm-‘) through the c&mm either under gravity or reduced pressure (maxunum 30 mmHg) The columns were washed Hrlth water (10 ml) followed by acetomtrde (4 ml) Fmally, the salbutamol was eluted from the column Hnth 99 5% methanol (6 ml)

L Howells et al /Anal Chwn. Acta 275 (1993) 275-278 277

The eluate was dned under mtrogen at 37°C and then redissolved m 0 5 ml PAS-gelatm buffer pnor to determination by enzyme immunoassay

Enzyme unmwwassay Salbutamol standards and three batches of 12

samples were analysed on each mlcrotltre plate usmg the enzyme immunoassay described by Sauer et al [15] A cahbratlon graph covermg the range from 10 to 5000 pg salbutamol per well was used to interpolate salbutamol concentration

LC clean-up Ahquots of the extract (200 ~1) were mjected

onto a 3 pm Hypersll ODS column (100 X 4 6 mm 1 d) with a 5 pm Lichrosorb RP18 guard column (5 x 3 mm 1 d > The mobile phase was 15% methanol 111 0 2% orthophosphorlc acid pumped at a flow-rate of 1 ml mm-’ and a fraction collected from 1 mm before the retention tnne of Salbutamol until 2 mm after The fraction was buffered to pH 7 f 1 with 11 ~1 of sodium hydroxide solution (10 M) and taken to dryness on a Gyrovap centrifugal evaporator (45”C, 3 h) It was redissolved m 100 ~1 of water and assayed by the enzyme unmunoassay

TABLE 1

Speclfictty and sensltmty of the method

RESULTS AND DISCUSSION

Survey of field samples The method was used to analyse 18 batches of

samples from the UK “Residues m Meat” pro- gramme The recovery of the radlolabel m these batches enabled estlmatlon of the reproduclblhty of the extraction procedure the mean recovery was 57% with a coefficient vanatlon of 8% (n = 14)

It was also possible to obtam an estunate of the vanabdlty of B/B, (%o) [tti 1s a measure of the enzyme labelled clenbuterol bound m the presence of competmg salbutamol (B) expressed as a percentage of that bound m the absence of competmg salbutamol (B,)] values for the nega- tive liver extracts m this population of field sam- ples (1 e , those not contammg salbutamol or any other cross-reactants) this was done by analysis of variance wlthm the batches followmg the re- moval of outhers (results outside three standard devlatlons from the batch mean) and was found to be 5 64% The B/B, (%o) values given by liver samples, forttiled to contam 1 pg kg- ’ salbuta- mol, were far enough outside this range for num- bers of false posmves and false negatives to be acceptable The mean difference between B/B,

B/B, (%) umts Specticity SensltMy below mean of tisue blanks a

No of tissue blanks per batch

No of tissue blanks per batch

1 2 3 4 1 2 3 4

1 0747 0778 0792 0800 0994 0998 0999 0999 2 0785 0819 0833 0841 0992 0997 0999 0999 3 0820 0855 0869 0876 0989 0996 0998 0998 4 0851 0885 0899 0906 0985 0994 0996 0997 5 0878 0911 0923 0930 0979 0991 0994 0995 6 0902 0932 0943 0949 0972 0987 0991 0993 7 0922 0949 0959 0%3 0963 0981 0986 0989 8 0938 0962 0971 0974 0952 0974 0980 0983 9 0952 0973 0979 0982 0939 0964 0972 0976 10 0963 0981 0986 0988 0922 0951 0960 0%5 11 0972 0987 0991 0992 0903 0914 0926 0933

’ B/B, (%) IS a measure of the enzyme labelled clenbuterol bound m the presence (B) of competmg salbutamol expressed as a percentage of that bound m the absence of competmg salbutamol

278 L HowelLs et al /Anal Chm Acta 275 (1993) 275-278

TABLE 2

Residues of salbutamol m bvers of treated animals [Ammals were tqlected (mtravenous) with salbutamol sulphate (Ventolm) 1 h pnor to slaughter]

Dose (pe kg-9 02 10 50 Salbutamol measured @g kg-‘) 15 16 4 5

(%) for the negative samples and fotifred sam- ples was shown to be 215%

Table 1 shows the speclfic~ty and sensitlvlty [16] obtamed when dtierent values below the blank control tissue were taken to defme the posltmes threshold Usmg these thresholds is hkely to underestnnate the sensltMty of the method because III practlse many posrtnre sam- ples wrll contam more than 1 pg salbutamol kg- ’ and would have lower B/B, (%o) values

Smce we msh to detect at least 99% of all positwe samples (sensltltmty = 0 99) the threshold value was set at 5 B/B, (%o) “units” below the mean tissue blank [I e., samples v&h B/B, (%o) values less than this are taken as positwe] Ac- cordmg to Table 1 this will give ruse to a false positrves rate of 12% (spe&icity of 0 88)

Liver samples from 3 ammals that had been dosed vvlth salbutamol were analysed and the resuIts are shown m Table 2

Conftnnatwn Samples with B/B, (o/o) values below the

threshold were repeated m a batch contammg four blank control tissues This enables the threshold to be set at 8 B/B, umts below the mean of these four tissue blanks and would theo- retically gore ftse to a false positives rate of only 25%

The very small number of samples which still appeared positwe after this treatment were sub- jected to the LC clean-up described above This reduces the posslblllty that clenbuterol or other mterfermg or cross-reacting compounds will be present durmg the EIA We demonstrated the separation of clenbuterol from the salbutamol by collecting the salbutamol fractton of an extract

contammg radlolabelled clenbuterol Only 0 1% of the radiolabel appeared m the salbutamol frac- tion

Following LC clean-up all suspect posltrves were found to be negative except the spiked samples and those contammg mcurred salbuta- mol

The method desctlbed has therefore proved to be reliable m routme use and has enabled the screening of several hundred samples of bovme and porcine liver for the presence of salbutamol Its effectiveness at dtierentlatmg negative sam- ples from those spiked at 1 pg kg-’ and from samples containing incurred residues has been demonstrated

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