The determination of phenylalanine in serum by mass fragmentography

Clin. Biochem. 6, 300-306 (1973)

THE D E T E R M I N A T I O N OF P H E N Y L A L A N I N E B Y MASS F R A G M E N T O G R A P H Y

IN S E R U M

W. E. PEREIRA, V. A. BACON, Y. HOYANO, R. SUMMONS and A. M. DUFFIELD

Department of Genetics, Stanford University, Medical Center, Stanford, Califor~ia, 94305, U.S.A. (Received January 15, i973)

CLBIA, 6 (4): 300-306 (1973) Clin. Biochem. Pereira, W. E., Bacon, V. A., Hoyano, Y., Summons, R. and Duffield, A. M. Dept. of Genetics, Stanford University Medical Centre, Stanford, California, 74305 U.S.A. THE DETERMINATION OF PHENYLALANINE IN SERUM BY MASS FRAGMENTOGRAPHY The technique of mass fragmentography has been applied to the determination of phenylalanine in normal and phenylketonuric serum. The method is specific and capable of detection and quantitation of submicrogram quantities of phenylalanine.

T H E TECHNIQUE OF MASS FRAGMENTOGRAPHY (1) h a s g a i n e d w i d e acc e p t a n c e as a q u a n t i t a t i v e a n a l y t i c a l tool f o r t h e d e t e r m i n a t i o n of subm i c r o g r a m q u a n t i t i e s of d r u g s , a n d d r u g m e t a b o l i t e s , in biological t i s s u e s a n d fluids. T h e m e t h o d h a s b e e n e x t e n d e d to v a r i o u s o t h e r n a t u r a l l y occurr i n g biological c o m p o u n d s such as n e u r o h u m o r a l t r a n s m i t t e r s , ( 2 , 3 ) m e t a b o l i t e s (4) a n d b i o c h e m i c a l i n t e r m e d i a t e s (5). I n t h e p r o c e s s of e x t e n d i n g t h e t e c h n i q u e of m a s s f r a g m e n t o g r a p h y (1) f o r t h e s i m u l t a n e o u s q u a n t i t a t i o n of up to 10 a m i n o acids p r e s e n t in biological e x t r a c t s we h a v e d e v e l o p e d a n e w specific, q u a n t i t a t i v e a n d reliable m e t h o d f o r t h e d e t e r m i n a t i o n (6-9) of p h e n y l a l a n i n e in s e r u m .

Correspondence:

A.M. Du.ffield.

D E T E R M I N A T I O N OF P H E N Y L A L A N I N E

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Fig. 1. Standard curve for the quantitative determination of phenylalanine in serum.

METHODS DL-Phenylalanine was obtained from Nutritional Biochemical Corporation (Ohio). L-Phenylalanine-ds [C~Ds*CD.~*CD(NH2)COOH] was supplied by Merck Laboratory Chemicals (New Jersey). 2,4-Pentanedione was obtained from Eastman Organic Chemicals (New York). Gas chromatography-mass spectrometry data were obtained with a Varian model 1200 gas chromatograph coupled by an all glass membrane separator to a F i n n i g a n 1015 quadrupole mass spectrometer interfaced to the ACME computer system of the Stanford University Medical School. GLC separations were carried out using a 6 foot coiled glass column (i.d. % in packed with 10% OV-17 on Gas Chrom Q (80-100 mesh). Helium flow rate (30 m l / m i n ) .

PROCEDURE

To a serum sample (0.3 ml) is added 25/~1 of the internal standard (Lphenylalanine-d~, 1 mg/ml in 0.01N HCI). The solution is deproteinized with alcohol (1 ml), centrifuged, and the supernatant evaporated to

302

PEREIRA

et al.

dryness. MeOH/HC1 (1 ml) is added and the reaction mixture refluxed for 20 minutes. The solution is evaporated to dryness, MeOH (1 ml) added and re-evaporated to dryness. The residue is dissolved in MeOH (10 /zl), and to it is added triethylamine (10 /~i) and 2,4-pentanedione (30 tti). The mixture is kept at room temperature for 4 hours, before injecting an aliquot (2 /~l) into the injector port of the gas chromatograph, which after one minute is programmed from 170 ° to 280 ° at 4 ° per minute. To each of five solutions containing 10 tLg, 20/~g, 25t~g, 30 t~g and 40/zg of phenylalanine respectively, was added a solution containing 25 /~g of L-phenylalanine-d,. The solutions were evaporated to dryness and derivatized by the above procedure. The derivatized sample was injected into the gas chromatograph under the conditions described above. The peak for the phenylalanine derivative eluted from the column at 243 ° (T, = 18 min). This procedure was used to construct a standard curve (Fig. 1) for the quantitation of phenylalanine. RESULTS

The derivatization procedure involves conversion of phenylalanine (I) into the methyl ester hydrochloride (II), and the enamine derivative (III)

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(lid The mass spectrum of the enamine-methyl ester derivative of phenylalanine (III) and phenylalanine-d, (IV) is shown in Figs. 2 and 3 respectively. DISCUSSION In the technique of mass fragmentography the mass spectrometer is used as a detector to monitor only specific pre-selected ions present in the effluent of a particular gas chromatographic peak. In the computer quadrupole mass spectrometer system used in the present work (11) up

DETERMINATION

OF P H E N Y L A L A N I N E

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Fig. 3. Mass spectrum of the enamine-methyl ester derivative (IV) of L-phenylalanine-ds.

to 25 pre-selected different ions m a y be monitored sequentially although by modifying the computer control program additional ions m a y be sampled. The computer records the intensity of the deuterated and nondeuterated ions as a function of time, after which quantitation can be

304

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DETERMINATION

OF PHENYLALANINE

305

TABLE 1 SERUM PI-IENYLALANINELEVELS IN PHENYLKETONURIC AND NORMAL PATIENTS Serum Phen,~lalanine mg ~/o Assay Patient

#1

Phenylketonuric 1 2 3 4

34.0 33.0 26.6 22.6

#2 32.3 32.7 26.2 23.6

Normal 1 2

2.7 1.3

2.5 1.25

achieved by calculation of the ratio of the peak areas for the deuterated and non-deuterated ion currents. Work is currently in progress to expand this technique for the simultaneous detection and quantitation of up to 10 amino acids present in both soil and biological extracts. Since the mass spectrum of the enamine derivative (III) of phenylalanine contains a molecular ion only two ions were monitored, m / e 261 (molecular ion of III) and m / e 269 (molecular ion of IV). Although the enamine methyl ester derivative of phenylalanine has been used in this procedure it may be replaced by other amino acid derivatives (e.g. N-TFA, O-n-butyl) in which case the system would monitor a pre-selected ion species in the mass spectrum of the deuterated and unlabeled compound. The reproducibility of the method for the quantitative measurement of serum phenylalanine levels in normal and phenylketonuric patients is summarized in Table 1. A typical total ion chromatogram of a phenylketonuric serum sample is shown in Fig. 4.

ACKNOWLEDGEMENT

This research was funded by the Planetology Program office, Office of Space Science, NASA Headquarters, under grant NGR-05-020-004.

306

PEREIRA et al. REFERENCES

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