Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

Analytica Chimica Acta, 237 (1990) 61-69 Elsevier Science Publishers B.V., Amsterdam

61

Development and validation of a gas chromatographi-mass spectrometric procedure for the identification and quantification of residues of chloramphenicol L.A. van Ginkel

*, H.J. van Rossum, P.W. Zoontjes, H. van Blitterswijk, A.P,J.M. de Jong and G. Zomer

National Institute

G. Ellen, E. van der Heeft,

of Public Health and Environmental Protection, Laboratory for Residue Analysis, 3720 BA Bilthoven (The Netherlands) (Received

7th March

P.O. Box I,

1990)

Abstract A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [37C12]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 pg kg-’ are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices. Keywords;

Chloramphenicol;

Urine;

Eggs; Biological

materials

Chloramphenicol (CAP) is a bacteriostatic compound with a broad spectrum of activity, frequently used in veterinary practice for therapeutic and prophylactic purposes. However, in humans CAP can cause serious health problems, e.g., aplastic anaemia [l], but there are no data indicating the minimum amount of residue which can induce these effects. Pending the outcome of further studies, in 1969 the FAO/WHO Expert Committee on Antibiotics recommended a zero tolerance in meat products [2]. In several countries the use of CAP is banned for laying hens and lactating cattle. For meat products in most countries maximum residue levels 0003-2670/90/$03.50

0 1990 - Elsevier

Science Publishers

of l-10 pg kg-’ have been set. In the U.S.A. there is a zero tolerance. For the detection of residues of CAP, numerous methods have been developed, both for screening and for confirmation purposes. These methods are based on almost any analytical technique used in residue analyses, e.g., immunoassays [3,4], liquid chromatography (LC) [5-131, gas chromatography (GC) [16-191 and polarography [20]. For purposes of identification by means of direct structure information, UV spectrophotometry (diode-array) [ll] and mass spectrometry (MS) have been used [14,17]. The clean-up and concentration steps also differ greatly. ImmunoaffinB.V.

62

L.A. VAN

ity chromatography, one of the latest tools in sample clean-up procedures for residue analyses [3], has been shown to be very promising [21-231. The objective of this study was to develop a method suitable for both qualitative and quantitative confirmation of CAP residues in urine, meat and eggs. For qualitative confirmation the European Communities (EC) have set identification criteria which have to be fulfilled in order to prove the presence of an analyte with sufficient reliability [24]. In case of CAP, when there are sometimes non-zero residue limits, there is an additional problem of confirmation of the concentration. For a method to be suitable under different situations, it was felt necessary to aim at a detection limit well below 1 pg kg-’ for eggs and muscle. Urine was added to the list of matrices investigated, since routine screening is frequently performed on this material. Based on the above, a method based on GC-MS with negative-ion chemical ionization (NCI) detection has been developed. For purposes of accurate quantification an isotopically labelled internal standard was synthesized which has been reported earlier [25]. Here the analytical procedures, the identification criteria used in the GC-MS part of the analysis and the method validation are reported. Details of the GC-MS detection of CAP using different conditions, e.g., different ionization techniques, will be reported elsewhere [26].

EXPERIMENTAL

a

Standards and chemicals All solvents, reagents and chemicals were of analytical-reagent grade from Merck (Darmstadt) unless stated otherwise. Standard chloramphenicol, (l)( - )-threo-2-dichloroacetamido-l-p-nitrophenylpropane-1,3-diol was purchased from Boehringer (Mannheim). The isotope-enriched in-

a Reference to a company and/or product is for purposes of information and identification only and does not imply approval or recommendation of the company and/or the product by the National Institute of Public Health and Environmental Protection, to the exclusion of others which may also be suitable.

GINKEL

ET AL.

ternal standard [ 37C1 ,]chloramphenicol was synthesized as described [25]. fl-Glucuronidase was a Sigma product purchased from Brunschwig Chemie (Amsterdam), containing /3-glucuronidase and sulphatase activity. The solvent used for LC analysis was ethanol-isooctane (3 : 97, v/v). Samples Urine samples from untreated and treated animals were obtained from the Institute for Animal Nutrition (IWO) (Lelystad, The Netherlands) and The Netherlands Organization for Applied Scientific Research (ILOB) (Wageningen, The Netherlands). Eggs from untreated and treated animals were obtained from the Spelderholt Centre for Poultry Research and Information (Beekbergen, The Netherlands; Dr. C.A. Kan) and muscle from a treated cow was a gift from the Bundesgesundheitsamt (Berlin, F.R.G.; Prof. Dr. D. Arnold). The samples from the survey were collected on behalf of the Dutch Veterinary Chief Inspectorate. Apparatus The instruments used were a Moulinette homogenizer, an Ultra-Turrax mixer (Janke und Kunkel, Staufer, F.R.G.), an RC-3 centrifuge (Sorvall) and a Rotavapor (Btichi). The LC system consisted of a Model 6000 solvent-delivery system, a WISP auto-injector, a Model 440 UV detector and an integrator (Data Module), all from Waters-Millipore, and a Redirat fraction collector (Pharmacia-LKB). A Chromguard (Chrompack Middelburg, The Netherlands) guard column (10 mm x 3.0 mm i.d.) and an analytical column packed (Column Packing Instrument, Shandon) with LiChrosorb Diol(5 pm) (Merck) (150 mm X 4.6 mm i.d.) were used. All GC-MS analyses were carried out on a Finnigan (San Jose, CA) Model 4500 quadrupole mass spectrometer combined with a Finnigan 9610 gas chromatograph containing a CP-Sil-19CB fused-silica column (25 m X 0.25 mm i.d.) with a film thickness of 0.25 pm (Chrompack). The end of the column was passed through the separator oven and inserted directly into the source of the mass spectrometer. Helium was used as the carrier gas at a column head pressure of 1.1 X lo5 Pa.

CC-MS

OF CHLORAMPHENICOL

RESIDUES

Splitless injections were made at an injection port temperature of 260 o C. The GC oven temperature was increased 1 min after injection ballistically from its initial temperature of 150 to 270 o C. The separator oven and transfer line temperatures were maintained at 260 and 270° C, respectively. The mass spectrometer was operated in the negativeion chemical ionization mode at a source temperature of 140 o C and with a filament emission current of 200 PA. Methane was used as the reagent gas with an ion-source pressure of 50 Pa. The electron energy was usually optimized between 70 and 90 eV. Data were acquired in the selected ion monitoring (SIM) mode, using a dwell time of 0.052 s per channel. Procedure for spiking Test samples of 10 g or 10 ml are spiked, after homogenization, with a solution (0.1 ml) of the internal standard. The amount of CAP added corresponds to approximately half the maximum concentration expected. The test sample is mixed thoroughly and allowed to stand for 30 min at room temperature or overnight at 4” C. Sample preparation Urine. The primary extract is prepared by Extrelut extraction, as previously used for the purification of aqueous extracts of muscle [ll]. To a lo-ml test sample, the pH of which was adjusted to 5.2 with acetic acid, 0.02 ml of glucuronidase and 0.5 ml of 2 mol 1-l acetate buffer (25.2 g acetic acid and 214.9 g sodium acetate trihydrate) (pH 5.2) are added. The sample is then hydrolysed for 2 h at 37OC. After cooling to room temperature, the sample is applied to an Extrelut cartridge (Merck) together with an additional 2 ml of water. The total aqueous phase is allowed to be absorbed in the column material for 15 min. CAP is extracted from the absorbed urine with two 25-ml portions of ethyl acetate. The combined eluate is evaporated to dryness under reduced pressure at 50” C and the residue is dissolved in 2 ml of methanol and transferred to a glass tube containing 5 ml of water. Two additional 2-ml portions of methanol are used to transfer the residue completely. The volume of the combined methanol-water phase is

63

reduced to ca. 4 ml under a stream of nitrogen in a water-bath at 50” C. This aqueous extract is further purified by solid-phase extraction (Sep-Pak C,, cartridge, Waters-Millipore). The cartridges are pretreated with 2 ml of methanol and 5 ml of water, after which the sample is applied. The cartridges are washed with 5 ml of water and consequently with 5 ml of methanol-water (10 : 90, v/v) and eluted with 5 ml of methanol-water (45 : 55, v/v). The eluate is evaporated to dryness under a stream of nitrogen in a water-bath at 50 o C and further purified by LC. Muscle and egg. From the laboratory sample a 200-g portion is homogenized. Eggs are broken and the contents are mixed to obtain a homogeneous suspension, whereas muscle is homogenized in a Moulinette homogenizer. From the test sample obtained a 10.0-g test portion is accurately weighed into a 250-ml centrifuge tube. To the homogenate 100 ml of ethyl acetate and 30 g sodium sulphate are added and the mixture is further homogenized for 1 min in an Ultra-Turrax mixer. The tubes are centrifuged (30 min, 1800 g). A 75-ml portion of the supematant is transferred to a round-bottomed flask (150 ml) and the solvent is evaporated in a Rotavapor under reduced pressure at a bath temperature of 50 o C. A Sep-Pak silica cartridge is pre-treated by flushing with 5 ml of acetonitrile-water (20 : 80, v/v), 5 ml of acetonitrile and 5 ml of dichloromethane. The cartridge is flushed with nitrogen. The residue of the extract is dissolved in 5 ml of dichloromethane and applied to the cartridge. The round-bottomed flask is rinsed with two additional 5-ml portions dichloromethane, all of which is applied to the cartridge. The cartridge is flushed with nitrogen until it contains no more dichloromethane. CAP is eluted with 5 ml of acetonitrile-water (20 : 80, v/v). The eluate is extracted three times with 1 ml of ethyl acetate. The combined extract is washed with 1 ml of water and evaporated to dryness in a water-bath at 50°C with a stream of nitrogen and purified further by LC. LC purification The dry residue of the extract LC eluent (0.3 ml). An aliquot

is dissolved in of 0.25 ml is

L.A. VAN

64

instructions of the supplier, drolysis [28].

CC-MS analysis The dry LC-purified extract is transferred with absolute ethanol to a derivatization vial and subjected to derivatization with 0.1 ml of N,O-bis(trifluoromethylsilyl)acetamide-1 W trimethylchlorosilane (Pierce) for 1 h at 60 o C. The reagent is then evaporated with a stream of nitrogen at 50 o C and the residue is dissolved in 0.025 ml of isooctane. GC-MS analysis is applied in the selected ion monitoring mode, recording the abundances of the ions of m/z 316, 378, 466,468 and 470.

Identification of CAP In residue analysis, the identification of the compound detected as the analyte is one of the most important aspects. For identification by GC-MS, the following criteria have been agreed upon [24]: the retention time for the compound detected has to be equal to that of the corresponding standard, with a tolerance of f5%; at least four diagnostic ions have to be monitored, having simultaneous responses at the retention time of the compound of interest; and the relative abundances (A) should differ by no more than +lO% from those for the corresponding standard. Figure 1 shows the NC1 mass spectrum of CAP. The two most abundant ions are the molecular ion at m/z 466 and the chlorine isotope ion at m/z 468 and the fragment ion and its isotope ion at m/z 376 and 378, respectively. Effectively the following criteria are used: the ratio A&A,,,

100

MIZ 5C

I1 50

62

I,

83

I

151 99 ll,9 133 1 I I1 I I I1 100 150

171,.186. 2p2 268 . 216225, .,.,L, " 21.2 ,. I I I I I I I I I I I 200 250 L66

loo-

376

503I.2

OT7_

3OL 322 292 , Lt., ,L., 300

Fig. 1. Structure

; L,

350 If". ,

350

of CAP and NC1 mass spectrum

(39L I.,

LlL , ,

hy-

RESULTS

Immunochemical procedures To validate the developed procedure at levels below 5 pg 1-i in urine, a radioimmunochemical procedure (RIA) was used [27]. For higher concentrations the Quik-Card (Environmental Diagnostics, Burlington, NC) was also used. This enzyme immunoassay was used according to the

L6

enzymatic

ET AL.

injected in the LC system and the CAP fraction is collected for 1 min, starting 0.5 min before the retention time of CAP. The fraction is evaporated to dryness in a water-bath at 50 o C under a stream of nitrogen.

0

after

GINKEL

L32 y,y

LOO

of CAP-diTMS.

L50 y" , L50

GC-MS OF CHLORAMPHENICOL

65

RESIDUES

(ri) has to be within 10% of the theoretical value of 0.76, the ratio A3,s/A376 has to be within 10% of the theoretical value of 0.70 and the ratio A&A,,, (rz)’ has to be within 10% of the value for the corresponding standard, determined experimentally under the same conditions as for the sample.

Analysis of urine from animals not treated with CAP Samples were analysed with both the RIA and the described GC-MS method. All samples (n = 20) showed an immunochemical response below 0.4 pg 1-i CAP equivalent, whereas CAP was not detected in the GC-MS analysis.

Quantification of CAP Figure 2 shows the NC1 mass spectrum for the internal standard. The two most abundant fragments are at m/z 470 and 380. As this standard contains isotopically pure 37C1, no isotope cluster ions are observed. For purposes of quantification, the molecular ion at m/z 470 is recorded in addition to the fragment ions mentioned above. For quantification the ratio A466/A~470 is calculated, where AC,,, is the corrected value of A,,, according to AC,,, = A,,, - O.l84A,,,. This correction is necessary since the chlorine isotope peak of the ion of m/z 466 contributes to the response at m/z 470. Linearity over the range O-20 pg 1-l is good (correlation coefficient 0.9992, intercept not deviating significantly from zero).

Analysis of urine from animals treated with CAP Seventeen samples from animals treated with CAP, obtained at different times after treatment, were analysed by the GS-MS method and the two immunochemical procedures (Table 1). The RIA responses were confirmed as being caused by CAP by GC-MS with responses ranging up from 0.3 pg 1-l. Samples containing more than 4 pg 1-i according to RIA were also judged positive with the Quik-Card. Repeatability and reproducibility The repeatability was tested for urine as the sample matrix in three different experiments at two CAP levels, with five replicates each time. The experiments were done by different technicians,

0

02N-@CH-CH-NH-?-CH37C12 I

HA Cti20H 151 119

o .46 I 50

58r

I

75 03 111 1’ ,9L 1 ,,, I I’ 100

I

135 1L5 161 i’ ’ I -I’ ‘r 150

177 166 1” “‘.~““‘i’

202 216225 ,J_ I”’ “I * “’ 200

.?$. 260 I I , 250 L '0

1007

TZ so-

380

3OL 322 270 288 Ou.; ..I.,Li. , IL 31L ,I, , 300

Fig. 2. Structure

of

360 3.46 398 ,A. , .,t.. , , .tlr , _.I. 350 LOO

[“Cl ,]CAP and NC1 mass spectrum

L16

L32 t_ . LL7.J.. I L 650

of [ 37Cl ,]CAP-diTMS

(internal

IL standard)

after negative

chemical

ionization.

66

L.A. VAN

TABLE

I

TABLE

GINKEL

ET AL,

2

Results obtained for 17 samples of urine with the RIA, enzyme immunoassay (Quik-Card) and GC-MS procedures (samples arranged according to their response in the RIA)

Repeatability (r) and reproducibility (R) calculated for urine and determined in three different experiments, five replicates each time (GC-MS method)

Sample No.

(Pg 1-l)

RIA

GC-MS

a

Quik-Card

Concentration

b

(Pg I-‘)

H141181 H140932 H140945 H140858 H140872 H140929 H144612 H141012 H141178 H144617 H146940 H141197 H144624 H141197

0.01 0.30 0.50 0.78 1.1 1.1 1.1 2.6 2.7 4.2 4.7 >lO >lO >lO

+ + + + + + + +

_ _

+ + + + +

+ + + + +

2.83 12.0

Within-assay variability

Between-assay variability

r

r.s.d. (W)

R

r.s.d. (%)

0.63 0.8

8.0 2.3

0.69 3.3

8.7 9.9

Using analysis of variance, the within-assay (groups) variance (sf) and the between-assay (groups) variance (s:) were calculated. From the within-assay variance the repeatability (r) can be

a + (GC-MS): all criteria for identification were fulfilled. b + (Qt.&-Card): the response of the Quik-Card was positive according to the instructions of the supplier.

calculated according to r = 2.8g. The betweenassay variance can be used as an estimate of the reproducibility (R), the time, chemicals and technicians being different but the laboratory being the same [R(L)]. R(L) was calculated according to

using different batches of chemicals and standards. The period between successive experiments was at least 1 month.

R(L) = 2.8@. In Table 2 the results are given for a high (12.0 pg 1-i) and a low level (2.83 pg 1-i). In addition, the relative standard deviations are given [r.s.d.(%) = (s/mean) X 1001. The results fulfil the EC criteria for reference methods [31].

M/Z

L--w

37600 050

?

-

I

1

37800

1760

a La

1780 a:5L

Fig. 3. Chromatograms

la00

9 00

(ion traces)

i a20

9:06

laL0

9.12

1860 9:18

for the five ions monitored

1660

9.2L

as recorded

1900 9 30

1920 SCAN 9 35 TIME

for a urine sample

containing

1 pg CAP I-’

CC-MS

OF CHLORAMPHENICOL

61

RESIDUES

Limit of detection Figure 3 shows the mass chromatograms of the five (fragment) ions as detected for a urine sample containing 1.0 pg CAP 1-i. This sample was prepared by dilution of the material used for estimating the repeatability and reproducibility with urine in which CAP was not detectable. Neither identification nor quantification at this level was a problem. In order to estimate the limit of detection (the lowest concentration at which all ions can be detected, with the ratios r,, r, and r, fulfilling the criteria), this sample was further diluted with blank material. Figure 4 shows the relationship between the calculated and measured concentrations as determined in two experiments. The regression lines are statistically identical. There is a small positive intercept of 0.03 f 0.01 the response IJg 1-l (mean f 1 s.d.). However, measured with the blank sample did not fulfil the criteria for identification. At levels > 0.1 pg 1-i all criteria could be fulfilled in all experiments performed at that level. Results for egg and muscle The procedure was evaluated for egg and muscle by diluting material, known to contain ca. 8 pg kg-‘, from animals treated with CAP with material from animals known not to be treated. The concentrations of CAP-containing material in the test

w ;

0.10

TABLE

3

Results (pg from treated CAP-containing material (W)

0 10 50 100 Av. s r.s.d. (%)

kg-‘) for eggs and muscle (CC-MS): material animals mixed with CAP-containing material Egg CAP diluted sample

Muscle CAP original sample

CAP diluted sample

CAP original sample

8.35 7.78 7.33

0.860 4.19 8.13

8.60 8.38 8.13

_ 0.835 3.89 7.33

_

7.82 0.51 6.5

8.37 0.24 2.8

sample were 0, 10, 50 and 100%. The results are summarized in Table 3. The material from untreated animals did not show a response fulfilling the identification criteria. All other samples, including samples with a CAP concentration below 1 pg kg-‘, showed responses fulfilling all the criteria. The quality of the chromatograms justifies the conclusion that the limits of detection for egg and muscle are of the same order as that for urine, i.e., 0.1 pg kg-‘. Results of a survey Urine samples from cattle, collected randomly in different slaughterhouses in The Netherlands (n = 100) were analysed for the presence of residues of several anabolic agents and veterinary drugs, including CAP. All samples were analysed with the RIA procedure. Figure 5 shows the frequency distribution of the immunochemical responses detected. The ten samples giving the highest immunochemical responses were analysed further with the Quik-Card procedure, and did not yield positive results. The two samples with the highest immunochemical responses (0.6 and 1.9 pg 1-l) were found positive by GC-MS.

E

DISCUSSION calculated

concentration

Fig. 4. Comparison of calculated and measured centrations in urine at levels below 0.5 pg I-’ Experiment 1; A, experiment 2.

kmb)

CAP con(ppb). +,

Methods to be used for the confirmation of CAP must be able to confirm both the identity and the amount. For confirmation of the identity

68

L.A. VAN

0

v4

N 0

1 0 II N 0

.J 0 2

: .J 0

:

L

:

x

m 0 r. 0

z

o

:

z

c _ N ;;‘,‘,,;‘& _ _

radlolmmunochemtcal

Fig. 5. Frequency Netherlands.

distribution

of RIA

results

(pg

”

”

r

r

_

m _

m _

K

_

c

-”

c

;

i

9

i

GINKEL

ET AL.

response

1-l)

for bovine

a set of criteria has been set by the EC [24]. For GC-MS, these criteria require the detection of at least four diagnostic ions with the correct response ratios. With NC1 one of the advantages is increased sensitivity for suitable components and the detection of the molecular ion. However, with respect to the confirmation, a disadvantage is the limited number of fragments. For CAP two abundant ions are produced, but both have a characteristic isotope cluster, mainly due to the presence of two chlorine atoms in the molecule. Two of the four ions monitored are therefore isotope ions. For accurate quantification, an internal standard is necessary for correction of losses during sample clean-up and for variations in the sensitivity of the detection system. Isotopically enriched internal standards are very suitable but their applicability is limited to RIA (tritiated internal standards) and GC-MS (stable isotope enriched internal standards). In the RIA procedure tritiated CAP was used as an internal standard [27], and in GC-MS [ 37C1,]CAP internal standard. Quantification with the latter standard resulted in highly precise results (low variability). As certified reference materials are not available it is difficult to estimate the accuracy. However, it is our belief that the method does not give a positive or nega-

urine

samples

(n = 100) obtained

during

a

in The

tive bias since there was good parallellism between the standards and samples, the results for diluted samples corresponded with the expected values, estimated based on analyses with different methods at higher concentrations, and no bias was shown for spiked blank samples. The method is therefore suitable for confirmation of the identity and concentration of CAP in urine, muscle and eggs at concentrations > 0.1 pg kg-‘. In view of the toxicological problems that can be caused by CAP and in spite of the fact that there are situations in which there is a zero tolerance, this limit of detection and determination is regarded as adequate for control purposes. Of course, the method is also suitable at the 10 pg kg-’ level. However, at this level simpler procedures are also available. Omitting the LC purification step from the present procedure gave good results at this level. No results for milk have been included. However, there are several good extraction procedures for milk that can be used as a sample preparation step for LC purification which allow comparable limits of detection and identification for this matrix [5,20,32]. For screening purposes the RIA method is suitable for urine with a limit of detection of 0.4 pg

CC-MS

OF CHLORAMPHENICOL

RESIDUES

1-i. However, the Quik-Card, with a limit of detection of ca. 5 pg l-l, can also be used. The two samples found ,positive by RIA and GC-MS but negative with the Quik-Card during the survey contained less than 5 pg 1-i. These urine concentrations correspond to concentrations in muscle below 1 pg kg-’ [29]. The limit of detection of the Quik-Card was also tested under reproducible conditions (seven Dutch laboratories). No false positives were observed and at levels of 6 pg 1-i and higher also no false negatives [30]. The combination of screening with the Quik-Card enzyme immunoassay of samples of urine, combined with confirmation of the identity and concentration by GC-MS, is a reliable strategy for effective control of residues of CAP. This investigation was made within project 388702 on behalf and for the account of the Dutch Veterinary Chief Inspectorate of Public Health.

REFERENCES 1 R.D. Wallerstein, Ph.K. Condit, C.K. Kasper, J.W. Brown and F.R. Morrison, J. Am. Med. Assoc., 284 (1969) 2045. 2 World Health Organization, 12th Report of the Joint FAO/WHO Expert Committee of Food Additives, WHO Technical Report Series, No. 430, FAO Nutrition Meeting Report Series, No., 45, FAO/WHO, Geneva, 1969. 3 C. van de Water and N. Haagsma, J. Chromatogr., 411 (1987) 415. 4 D. Arnold and A. Somogyi, J. Assoc. Off. Anal. Chem., 68 (1985) 984. 5 C. van de Water, N. Haagsma, P.J. van Kooten and W. van Eden, Z. Lebensm.-Unters.-Forsch., 185 (1987) 202. 6 U.R. Tjaden, D.S. Stegehuis, B.J.E.M. Reeuwijk, H. Lingeman and J. van der Greef, Analyst, 113 (1988) 171. 7 M.F. Pochard, G. Burger, M. Chevalier and E. Gleizes, J. Chromatogr., 409 (1987) 315. 8 G. Knupp, G. Bugl-Kreickmann and C. Commichau, Z. Lebensm.-Unters.:Forsch., 184 (1987) 390. 9 M.K. Aravind, J.N. Miceli, A.K. Dore and R.E. Kauffman, J. Chromatogr., 232 (1982) 461. 10 D.J. Berry, J. Chromatogr., 385 (1987) 337. 11 H.J. Keukens, W.M. Beek and M.M.L. Aerts, J. Chromatogr., 352 (1986) 445.

69 12 N. Haagsma, C. Schreuder and E.R. Rensen, J. Chromatogr., 363 (1986) 353. 13 J. Boisseau, Ann. Rech. Vet., 16 (1985) 15. 14 P. First, Chr. Kruger, H.A. Meemken and W. Groebel, Dtsch.-Lebensm.-Rundsch., 84 (1988) 108. 15 W. Loescher, 0. Dornheim and F. Mueller, Arch. Lebensmittelhyg., 36 (1985) 109. 16 B. Bergner-Lang and M. Kaechele, Dtsch. Lebensm.Rundsch., 81 (1985) 278. 17 F.A. Bories, J.C. Peleran and J.M. Wal, J. Assoc. Off. Anal. Chem., 66 (1983) 1521. 18 J.R. Nelson, K.F.T. Copeland, R.J. Forster, D.J. Campbell and W.D. Black, J. Chromatogr., 276 (1983) 438. 19 R. Malisch, Z. Lebensm.-Unters.-Forsch., 184 (1987) 467. 20 W.G. de Ruig and H. Hooijerink, Neth. Milk Dairy J., 39 (1985) 155. 21 L.A. van Ginkel, R.W. Stephany, H.J. van Rossum, H.M. Steinbach, G. Zomer, E. van der Heeft and A.P.J.M. de Jong, J. Chromatogr., 489 (1989) 11. 22 L.A. van Ginkel, H. van Blitterswijk, P.W. Zoontjes, D. van den Bosch and R.W. Stephany, J. Chromatogr., 445 (1988) 385. R. Schilt, A.R.M. Hamers, F.A. Huf, A. 23 W. Haasnoot, Fajam, R.W. Frei and U.A.Th. Brinkman, J. Chromatogr., 489 (1989) 157. of the European Communities, Off. J. Eur. 24 Commission Commun., 223 (1987) 18. J.F.C. Stavenuiter, G. van de 25 G. Zomer, M. Jacquemijns, Werken, A. de Jong, L.A. van Ginkel, H.J. van Rossum, R. den Hartog and G. Engelsman, in T.A. Baillie and J.R. Jones (Eds.), Proceedings of the Third International Symposium, Innsbruck, Austria, Synthesis and Applications of Isotopically Labelled Compounds, Elsevier, Amsterdam, 1989, pp. 777-780. 26 E. van de Heeft, A.P.J.M. de Jong, L.A. van Ginkel, H.J. van Rossum and G. Zonen, Biomed. Environm. Mass Spectrom., submitted for publication. 27 L.A. van Ginkel, H.J. van Rossum, H. van Blitterswijk and P.W. Zoontjes, RIVM Report No. 388702 001, Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands, April 1987. 28 J.F.M. Nouws, F. Reek, M.M.L. Aerts, M. Beekman and J. Laurensen, Arch. Lebensmittelhyg., 38 (1987) 9. 29 A.K. Boertz, D. Arnold and A. Somogyi, Z. Emlhrungswiss., 24 (1985) 113. 30 L.A. van Ginkel and H.J. van Rossum, RIVM Report No. 388702 004, Rijksinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands, July 1989. 31 Commission of the European Communities, Off. J. Eur. Commun., 351 (1989) 39. 32 D.P. Schwartz and F.E. McDonough, J. Assoc. Off. Anal. Chem., 67 (1984) 563.