Z Lebensm Unters Forsch (1992) 195:566--568

Zeitschrift for

9 Springer-Verlag 1992

Original paper Immunoaffinity-chromatography purification of salbutamol in liver and HPLC-fluorometric detection at trace residue level Jean-Marie Degroodt, Brigitte Wyhowski de Bukanski, and Sarah Srebrnik Minist~re de la Sant~ Publique et de l'Environnement, 14 rue J. Wytsman, B-1050 Bruxelles, Belgium Received May 21, 1992

Immunoaffinit~itschromatographisehe Reinigung von Salbutamol in Leber und Riickstandsbestimmung mittels HPLC und fluorimetrischer Detektion Zusammenfassung. Eine Methode, die Immunoaffinitatsund Hochleistungsfliissigchromatographie verbindet, ist ffir die Analyse yon Salbutamol in Leber entwickelt worden. Eine niedrige Bestimmungsgrenze von 1 ~tg/kg Leber wurde erreicht. Salbutamol wurde mit 0,01 mol/L Salzs/iure freigesetzt und durch Immunoaffinitfitschromatographie gereinigt. Die Proben wurden dutch Flfissigchromatographie auf einer C18 I~-Bondapak-S~ule analysiert. Ein Fluorimeter wurde fiir die Detektion yon Salbutamol benutzt. Ausbeuten von 67 bis 80% konnten erreicht werden. Summary. A method combining immunoaffinity-chromatography (IAC) and high-performance liquid chromatography (HPLC) for the analysis o f Salbutamol in liver with a low quantification limit of 1 gg/kg has been developed. Salbutamol was extracted with 0.01 mot/L HC1 and purified by IAC. The samples were analysed on a liquid chromatograph fitted with a C18 g-Bondapak column. A fluorometer was used for the detection of salbutamol. Recoveries of 67-80% could be obtained.

tography-mass spectrometry has been proposed [4] as well as H P L C analytical methods for the detection of salbutamol in plasma [5, 6], tablets [7] and H P L C in combination with enzyme immunoassay in urine [8]. Only little is known about specific purifications and determinations of salbutamol with H P L C in liver or other edible tissues. The most suitable sample clean-up for a small amount of a compound that has to be isolated from a complex matrix seems to be immunoaffinity chromatography (IAC). This technique has been used with great success for anabolic residue analysis in meat and bovine urine [9, 10], for chloramphenicol in muscles [11], for fl-agonists in premixes, finished feed, and urine [12] and for salbutamol in human plasma [13]. We propose an I A C - H P L C method with fluorometric detection for the determination o f salbutamol in liver, which eliminates all interferences proceeding from this complex matrix.

Materials and methods Apparatus. Liver was minced by a Moulinette S and an ultrasonic

[1-(4-hydroxy-3-hydroxymethylphenyl)-2-

bath Bransonic 5200 from Branson (Conn., USA) was used to liberate salbutamol from the matrix. Centrifugations were achieved by a J-21 B centrifuge from Beckman (Calif., USA) and evaporations of the eluent by simultaneous use of nitrogen and a Reacti-Therm heating module from Pierce (II1., USA) in which the tubes were immersed. HPLC analyses were performed with a 5000 liquid chromatograph from Varian (USA). Salbutamol was detected with an LS-4 fluorescence spectrometer from Perkin-Elmer (USA) (excitation wavelength, 230 nm; emission wavelength, 309 nm; slits, 15 and 20; recorder scale, 2). The chromatograms were registered with a A-25 Varian recorder (speed, 2 mm/min).

tert-butyl-amino-ethanol] is known as a fle-adrenergic

Solvents and reagents. All reagents were for analytical purposes.

Introduction Salbutamol

agonist and illegally used as a growth promoter [1, 2]. Recently we described an identification method of salbutamol in liver [3] purified on reversed-phase octadecyl C18 columns. To measure the drug quantitatively with H P L C at a low level, an extract without interfering compounds is necessary and the above mentioned purification is not sufficient. Enzymatic digestion of liver and gas chromaCorrespondence to: J.-M. Degroodt

Acetic acid glacial, ethanol absolute (pro analysi), potassium dihydrogen phosphate GR (KHzPO4) and disodium hydrogen phosphate-2-hydrate GR (NazHPO4 • 2HzO) were supplied by Merck (Darmstadt, Germany), phosphoric acid 85% by Carlo Erba (Milan, Italy), acetonitrile (HPLC grade) by Labscan (Dublin, Ireland), 37% HC1 and sodium azide (NAN3) by UCB (Bruxelles, Belgium) and NaC1 by RPL (Louvain, Belgium). - The IAC gel used (antiserum, antisalbutamol HMS-BSA), specific for flz-agonists, was obtained from the Laboratory of Hormonology (Marloie, Belgium). The gel has to be stored at 4~ C.

567

Solutions. For the extraction a 0.01 mol/L HC1 solution was used, and for the purification, a phosphate buffer 0.04 mol/L + 0.9% NaC1, pH 7.0. This phosphate buffer was used to prepare a 0.1% sodium azide buffer, pH 7.5. An elution solvent was prepared by mixing a solution of 0.5 mol/L NaC1 in 0.1 mol/L acetic acid and ethanol (50+50; v/v), pH 3.5. The rinsing solution consisted of ethanol/water (80 + 20; v/v). - The mobile phase used for HPLC was a mixture of acetonitrile and distilled water containing 0.15 % phosphoric acid (3 + 97; v/v). Using this very small quantity of acetonitrile, solvent and small amounts of impurities were eluted far before salbutamol. Standard and standard solutions. Salbutamol was purchased from Sigma Chemical Comp. (St. Louis, Mo, USA).

Purification procedure of salbutamolfrom liver by [AC. A general guideline for the use of the gel and the columns was given by the Laboratory of Hormonology where we bought the gel. The IAC gel may be very easily destroyed and should never be dry. It is therefore important to follow strictly the instructions. Figure 1 summarizes the use of the IAC columns including the modifications that we added.

Chromatography. For the analyses, 100 Ixl of the standard and 50 gl of each extract were injected at room temperature into the chromatograph fitted with a Cls g-Bondapak reversed-phase column. The mobile phase had a flow rate of I ml/min and salbutamol was detected by fluorescence.

H

HO-

Extraction procedure ofsalbutamolfrom liver. Minced liver (4 g) was mixed with 20 ml 0.01 mol/L HC1 in a centrifuge tube and immersed in an ultrasonic bath for 20 rain. After centrifugation at 9000 rpm in a Beckman J-21 B centrifuge for 15 min the supematant was transferred into another centrifuge tube, adjusted to pH 7.0 with NaOH solution and again centrifuged at 9000 rpm for 15 min.

- CHCH2NHC(CHa)a

CH20H Scheme 1. Structural formula ofsalbutamol

Results and discussion The standard solution contained 50 gg salbutamol/ml H20 and had to be stored in a refrigerator. The working standard solution was a solution of 0.2 gg salbutamol/ml 0.01 mol/L HC1. This solution was used to fortify the samples and to prepare a standard curve. The working standard solution was diluted ten times in 0.01 mol/L HC1 before use as a standard for HPLC analyses.

Columns. For the immunoaffinity purification procedure 10-ml Econocol columns and two-way stop-cocks from Bio-Rad (Nazareth, Belgium) were used. The HPLC column was a ix-Bondapak C18, 10 ~tm column (3.9 mm x 300 mm) from Millipore-Waters (USA).

Column + 1 ml IAC gel vial rinsed 3 x with 1 ml phos. buffer + 2 x 5 ml phos. buffer + 2 x 5 ml water + extract + 5 x 5 ml water + 3 ml elution solvent

~ evaporation residue + 0.2 ml water (ready to be injected) Use of the columnthe samedav Column + 2 x 5 ml rinsingsolution + 2 x 5 ml water + 2 x 5 ml phos. buffer + 2 x 5 ml water + extract

Storage of the columnat 4~

I c~176

I

+ 2 x 5 ml rinsing solution + 2 x 5 ml water + 2 x 5 ml sodiumazide buffer Use of the columnafter storage at 4~ I

Column + 2 x 5 ml water + extract

Fig. 1. Use ofimmunoaffinity-chromatography (IAC) columns

1

Residues o f fl-agonists i n edible a n i m a l tissues are n o t allowed. It has been s h o w n [14] t h a t the eye is the m o s t acc u m u l a t i n g tissue for fi-agonists in bovines. C o n c e r n i n g edible a n i m a l tissues, a collaborative study between the M i n i s t r y o f A g r i c u l t u r e a n d o u r l a b o r a t o r y showed t h a t c l e n b u t e r o l is m o s t l y a c c u m u l a t e d in the liver ( u n p u b lished data). T h u s we chose liver as the e x t r a c t i o n m a t e rial for s a l b u t a m o l . T h e directive 8 6 / 4 6 9 / E C C [1] imposes a surveillance p r o g r a m m e for residues o f salbutam o l in liver t h a t requires q u a n t i f i c a t i o n o f positive samples. T h e extraction step with 0.01 m o l / L HC1 was very simple. C o n c e r n i n g the p u r i f i c a t i o n step some c o m m e n t s are useful. I m m u n o a f f i n i t y - c h r o m a t o g r a p h y is a very selective m e t h o d . The i m m u n o a f f i n i t y gel used is specific for fie agonists. T o avoid d e g r a d a t i o n it has to be stored in the presence o f a bacteriostatic agent, such as 0.1% N a N 3 or 0.2% m e r t h i o l a t e at 4 ~ C. A n I A C c o l u m n m a y be used u p to ten times w i t h o u t a r e d u c t i o n i n recoveries, b u t the capacity o f the gel is limited to 50 ng. T h a t m e a n s , if a sample c o n t a i n s m o r e t h a n 10 gg s a l b u t a m o l / k g liver, less t h a n 4 g e x t r a c t i o n m a t e r i a l has to be used, or o n l y a fraction o f the extract m a y be applied to the c o l u m n . It is w o r t h m e n t i o n i n g also that the flow rate,was very low, _+ 8 d r o p s / r a i n with fully o p e n e d stop-cocks, b u t this time was necessary to allow b i n d i n g b e t w e e n s a l b u t a m o l a n d the a n t i b o d y . The H P L C analyses o f s t a n d a r d s resulted i n a linear response for s a l b u t a m o l w i t h i n a range o f 0.2 to 10 ng,

Table 1. Recovery of salbutanol in liver by HPLC and fluorometric detection Salbutamol added (gg/kg)

Mean of recovery (n=5) (~g/kg)

Standard deviation

1.25 2.50 5.00

1.00 1.68 3.37

0.09 0.15 0.30

Coefficient of variation (%)

Recovery

9.00 8.93 8.90

80 67 67

(%)

568 peak height of the sample with that of the standard. The result obtained was corrected by taking into account the recovery of the fortified sample. Positive samples were extracted twice again to obtain a mean of at least three results. On H P T L C plates salbutamol can be detected as a blue spot after derivatization [3]. This alternative method was used to confirm the results and to exclude false positive results.

70~

50 m o Q. m

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Conclusion

30

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,

a

c

The immunoaffinity-chromatography purification procedure allowed the detection and quantification of salbutamol. Excellent sensitivity and selectivity were achieved by H P L C and fluorescence spectroscopy.

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Acknowledgement. We wish to thank Mrs. L. Vifquin for technical

10

assistance.

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References

Time(min)

Fig.2a-c. Chromatograms obtained for a a salbutamol standard solution (2 ng/lO0 ~tl), b a blank liver sample and c a positive liver sample (3 ~tg salbutamol/kg) which corresponds to the capacity o f the I A C columns. The correlation coefficient and the confidence interval were 0.9998 and _+2.05 respectively. Table 1 summarizes the recoveries, standard deviations and coefficients o f variation using the experimental conditions indicated. The results were obtained by adding three different amounts o f salbutamol (1.25, 2.5 and 5 gg/kg liver). Each result is the m e a n of five different extractions. The recoveries varied f r o m 67 to 80%. The quantification limit of salbutamol which still allows a very good reproducibility was 1 ug/kg liver. The absolute detection limit o f salbutamol was 100 pg (recorder scale of the fluorometer: 12). Figure 2 shows a c h r o m a t o g r a m of a standard solution (2 ng salbutamol/100 gl), a blank liver sample and a positive liver sample containing 3 gg salbutamol/kg. Salbutamol was detected by fluorescence, which is k n o w n to be sensitive and selective, interference f r o m impurities having been avoided. The retention time for salbutamol was 10.5 min. The a m o u n t o f salbutamol extracted f r o m a positive liver sample was calculated by comparing the

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